Enhanced stem cell composition

ABSTRACT

The invention provides improved methods for cell therapy. In particular, the invention provides therapeutic compositions of enhanced hematopoietic stem and progenitor cells having improved engraftment and homing properties, and methods of making the therapeutic compositions. The invention further provides methods of improving the efficacy of hematopoietic stem and progenitor cell transplantation including transplanting the therapeutic composition to subjects in need of hematopoietic system reconstitution.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US2012/066987 filed Nov. 29, 2012,which application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/566,492, filed Dec. 2, 2011, each ofwhich is incorporated herein by reference in its entirety.

BACKGROUND

1. Technical Field

The invention relates to enhanced hematopoietic stem and progenitorcells and therapeutic compositions comprising the enhanced cells. Theinvention also relates to methods of making the enhanced hematopoieticand progenitor cells and therapeutic compositions and methods of usethereof, including use for reconstituting the hematopoietic system of anindividual and treating conditions and diseases associated withischemia.

2. Description of the Related Art

The goal of regenerative medicine is to maintain, improve or evenrestore the function of damaged or diseased cells, tissues, and organs.One way that regenerative medicine aims to revolutionize the practice ofmedicine is to employ cell-based therapeutics to treat patients.However, for the promise of cell-based therapeutics to be fullyrealized, the therapeutic cells should be well-tolerated when introducedinto a patient, the cells should also migrate or “home” to sites wheretherapy is needed, and the cells should be capable of providing thetherapy desired. The art has attempted to employ stem cell- andprogenitor cell-based therapeutics but has met with little, if any,success in a human clinical setting.

One area of regenerative medicine that would benefit from improvedcell-based therapeutics are stem cell transplants, e.g., bone marrowtransplants and hematopoietic stem cell transplants to treat variousgenetic diseases, cancers, and degenerative disorders. According to theNational Marrow Donor Program® (NMDP), an estimated 45,000 to 50,000hematopoietic cell transplants are performed annually worldwide to treatpatients with life-threatening malignant and non-malignant diseases.However, bone marrow transplantation has numerous drawbacks: bone marrowdonation is painful, at times it is difficult and time consuming, andoften not possible, to find HLA donor matched tissue; and allogeneictransplants are associated with a significant incidence ofgraft-versus-host-disease (GVHD). Moreover, although allogeneichematopoietic stem cell transplants have been performed using moreeasily obtainable umbilical cord blood, cord blood transplants stillhave a risk of GVHD. Other drawbacks to existing methods of cord bloodtransplants, include fewer numbers of transplantable cells and deficienthoming and engraftment of donor cells, both of which put the patient athigh risk for life threatening infections. In addition, cord bloodtransplants generally have all the same risks as marrow and peripheralblood transplants.

Numerous approaches have been tried to expand the number of humanhematopoietic stem and progenitor cells in cord blood within isolatedgrafts in ex vivo settings, to reduce the incidence of GVHD, or toincrease the ability of the cells to home and engraft, but these effortshave had limited success.

Another area of regenerative medicine that would benefit from improvedcell-based therapeutics is the treatment of tissue damaged by ischemia.Disruption of blood flow to tissues and organs is known as ischemia. Theviability of cells, tissues, and organs in the human body depends onadequate blood flow. Adequate blood flow provides cells with oxygen,glucose, and much needed nutrients that are important for the regulationof cellular physiology and metabolism. Ischemia can be acute or chronic.Both acute and chronic forms of ischemia result in the loss of adequatenutrients to the cells, and if prolonged, will result in hypoxic and/oranoxic conditions. If the ischemia is left untreated, the cells mayundergo necrosis or apoptosis, thereby jeopardizing the integrity andhealth of the tissue or organ.

Ischemia affects millions of patients in the United States each year.Ischemia is caused by a virtually limitless variety of geneticconditions, environmental insults, traumatic injury, or surgicalinterventions. The most common types of ischemia patients suffer frominclude, but are not limited to cerebral ischemias, spinal cordinjuries, cardiovascular ischemias, limb ischemias, intestinalischemias, dermal ischemias (e.g., burns and frostbite wounds) andischemias resulting from medical and surgical procedures, including, butnot limited to organ transplants, and skin grafts.

Currently, resolution of acute and chronic ischemia requires restorationof tissue perfusion and blood flow often using surgical means, whichfurther places patients as risk for ischemic tissue damage. Restorationof blood flow after a period of ischemia can actually be more damagingthan the ischemia. Reintroduction of oxygen causes a greater productionof damaging free radicals as well as allowing, via removal of theextracellular acidotic conditions, influx of calcium and thus calciumoverloading. Overall this results in reperfusion injury which can resultin potentially fatal cardiac arrhythmias, also necrosis can be greatlyaccelerated. Other existing treatments that address ischemic tissueinclude hyperbaric oxygen, intravenous thrombolytics, anti-inflammatoryagents, and local application of angiogenesis promoters. However, thesetreatments have generally met with limited success, if any.

Thus, many of the cell-based compositions and materials used inregenerative medicine are currently cost-prohibitive, inefficient,and/or unsafe. Other significant shortcomings for the use of stem cell-and progenitor cell-based therapeutic in regenerative medicine are thelack of technologies available to control stem cell proliferation,mobility, or to direct the stem cell, e.g., homing, to the particularniche or tissue where the therapy is needed. The end result is thatcell-based therapeutics are not considered a realistic treatment optionfor those in need of regenerative medicine.

Accordingly, there is a substantial need in the art for improvedcell-based therapeutics that are expandable, that are able to home tosites in the patient where therapy is desired, and that are able toprovide a persistent therapeutic benefit. The present inventionaddresses these needs and offers other related advantages.

SUMMARY OF THE INVENTION

The invention generally provides novel cell-based compositions withimproved therapeutic properties. In one embodiment, the presentinvention contemplates, in part, a human hematopoietic stem orprogenitor cell comprising a hematopoietic stem or progenitor cell thathas been contacted ex vivo with one or more agents that increase CXCR4gene expression in the cells and gene expression of CXCR4 is increasedat least about 30 fold in the contacted hematopoietic stem or progenitorcell compared to non-contacted hematopoietic stem or progenitor cells.

In a particular embodiment, the one or more agents comprises (i) one ormore prostaglandin pathway agonists; and (ii) one or moreglucocorticoids.

In a certain particular embodiment, the prostaglandin pathway agonistcomprises a compound that selectively binds the PGE₂ EP₂ or PGE₂ EP₄receptor.

In a further particular embodiment, the prostaglandin pathway agonist isselected from the group consisting of PGE₂, dmPGE₂, 15(S)-15-methylPGE₂, 20-ethyl PGE₂, and 8-iso-16-cyclohexyl-tetranor PGE₂.

In another particular embodiment, the prostaglandin pathway agonistcomprises PGE₂, or a PGE₂ analogue or derivative.

In an additional particular embodiment, the prostaglandin pathwayagonist comprises 16,16-dimethyl PGE₂.

In a certain embodiment, the glucocorticoid is selected from the groupconsisting of medrysone, alclometasone, alclometasone dipropionate,amcinonide, beclometasone, beclomethasone dipropionate, betamethasone,betamethasone benzoate, betamethasone valerate, budesonide, ciclesonide,clobetasol, clobetasol butyrate, clobetasol propionate, clobetasone,clocortolone, cloprednol, cortisol, cortisone, cortivazol, deflazacort,desonide, desoximetasone, desoxycortone, desoxymethasone, dexamethasone,diflorasone, diflorasone diacetate, diflucortolone, diflucortolonevalerate, difluorocortolone, difluprednate, fluclorolone, flucloroloneacetonide, fludroxycortide, flumetasone, flumethasone, flumethasonepivalate, flunisolide, flunisolide hemihydrate, fluocinolone,fluocinolone acetonide, fluocinonide, fluocortin, fluocoritin butyl,fluocortolone, fluorocortisone, fluorometholone, fluperolone,fluprednidene, fluprednidene acetate, fluprednisolone, fluticasone,fluticasone propionate, formocortal, halcinonide, halometasone,hydrocortisone, hydrocortisone acetate, hydrocortisone aceponate,hydrocortisone buteprate, hydrocortisone butyrate, loteprednol,meprednisone, 6a-methylprednisolone, methylprednisolone,methylprednisolone acetate, methylprednisolone aceponate, mometasone,mometasone furoate, mometasone furoate monohydrate, paramethasone,prednicarbate, prednisolone, prednisone, prednylidene, rimexolone,tixocortol, triamcinolone, triamcinolone acetonide and ulobetasol.

In a further certain embodiment, the glucocorticoid is medrysone.

In an additional certain embodiment, the stem or progenitor cells havebeen contacted with at least one agent for a time of at least about onehour.

In another certain embodiment, the stem or progenitor cells have beencontacted with at least one agent for a time of about one hour to abouttwenty-four hours.

In another certain embodiment, the stem or progenitor cells have beencontacted with at least one agent for a time of about one hour to abouttwelve hours.

In another certain embodiment, the stem or progenitor cells have beencontacted with at least one agent for a time of about one hour to aboutsix hours.

In a particular certain embodiment, the stem or progenitor cells havebeen contacted with at least one agent for a time of about two hours toabout six hours.

In a further embodiment, the stem or progenitor cells have beencontacted with at least one agent for a time of about two hours to aboutfour hours.

In an addition further embodiment, the cell is obtained from bonemarrow, umbilical cord blood, mobilized peripheral blood, Wharton'sjelly, placenta, or fetal blood.

In one embodiment, the present invention contemplates, in part, acomposition, e.g., a therapeutic composition, comprising a population ofcells comprising human hematopoietic stem or progenitor cells whereinthe hematopoietic stem or progenitor cells have been contacted ex vivowith one or more agents that increase CXCR4 expression in the humanhematopoietic stem or progenitor cells and gene expression of CXCR4 isincreased at least about 30 fold in the contacted hematopoietic stem orprogenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In an additional embodiment, the one or more agents comprises (i) one ormore prostaglandin pathway agonists and (ii) one or moreglucocorticoids.

In a particular additional embodiment, gene expression of CXCR4 isincreased by at least about 40 fold in the contacted hematopoietic stemor progenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In a particular additional embodiment, gene expression of CXCR4 isincreased by at least about 50 fold in the contacted hematopoietic stemor progenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In a particular additional embodiment, gene expression of CXCR4 isincreased by at least about 60 fold in the contacted hematopoietic stemor progenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In a particular additional embodiment, gene expression of CXCR4 isincreased by at least about 70 fold in the contacted hematopoietic stemor progenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In a particular additional embodiment, gene expression of CXCR4 isincreased by at least about 80 fold in the contacted hematopoietic stemor progenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In a particular additional embodiment, gene expression of CXCR4 isincreased by about 40 to about 80 fold in the contacted hematopoieticstem or progenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In a particular additional embodiment, gene expression of CXCR4 isincreased by about 50 to about 80 fold in the contacted hematopoieticstem or progenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In a particular additional embodiment, gene expression of CXCR4 isincreased by about 60 to about 80 fold in the contacted hematopoieticstem or progenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In certain additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by about 30, about 40, about 50, about60, about 70, or about 80 fold in the contacted hematopoietic stem orprogenitor cells compared to non-contacted hematopoietic stem orprogenitor cells and wherein gene expression of one or more genesselected from the group consisting of: hyaluronan synthase 1 (HAS1),GTP-binding protein GEM (GEM), dual specificity protein phosphatase 4(DUSP4), amphiregulin (AREG), Nuclear receptor related 1 protein(NR4A2), renin (REN), cAMP-responsive element modulator (CREM),collagen, type I, alpha 1 (COL1A1), and Fos-related antigen 2 (FOSL2) isincreased by at least about two fold, about three fold, about four fold,about five fold, about ten fold, about twenty fold, about thirty fold,about forty fold, or about fifty fold in the treated stem or progenitorcells compared to non-contacted stem or progenitor cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of one or more genes selected from the groupconsisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1, FOSL2is increased by at least about two fold in the treated stem orprogenitor cells compared to non-contacted cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of one or more genes selected from the groupconsisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1, FOSL2is increased by at least about three fold in the treated stem orprogenitor cells compared to non-contacted cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of one or more genes selected from the groupconsisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1, FOSL2is increased by at least about five fold in the treated stem orprogenitor cells compared to non-contacted cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of one or more genes selected from the groupconsisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1, FOSL2is increased by at least about ten fold in the treated stem orprogenitor cells compared to non-contacted cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of two or more genes selected from the groupconsisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1, FOSL2is increased by at least about two fold in the treated stem orprogenitor cells compared to non-contacted cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of two or more genes selected from the groupconsisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1, FOSL2is increased by at least about three fold in the treated stem orprogenitor cells compared to non-contacted cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of two or more genes selected from the groupconsisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1, FOSL2is increased by at least about five fold in the treated stem orprogenitor cells compared to non-contacted cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of two or more genes selected from the groupconsisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1, FOSL2is increased by at least about ten fold in the treated stem orprogenitor cells compared to non-contacted cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of three or more genes selected from thegroup consisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1,FOSL2 is increased by at least about two fold in the treated stem orprogenitor cells compared to non-contacted cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of three or more genes selected from thegroup consisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1,FOSL2 is increased by at least about three fold in the treated stem orprogenitor cells compared to non-contacted cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of three or more genes selected from thegroup consisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1,FOSL2 is increased by at least about five fold in the treated stem orprogenitor cells compared to non-contacted cells.

In other additional embodiments, contacted hematopoietic stem orprogenitor cells comprise a gene expression signature wherein geneexpression of CXCR4 is increased by at least 30 fold in the contactedhematopoietic stem or progenitor cells compared to non-contacted cellsand wherein gene expression of three or more genes selected from thegroup consisting of: HAS1, GEM, DUSP4, AREG, NR4A2, REN, CREM, COL1A1,FOSL2 is increased by at least about ten fold in the treated stem orprogenitor cells compared to non-contacted cells.

In a certain additional embodiment, the prostaglandin pathway agonistcomprises a compound that selectively binds the PGE₂ EP₂ or PGE₂ EP₄receptor.

In a further additional embodiment, the prostaglandin pathway agonist isselected from the group consisting of PGE₂, dmPGE₂, 15(S)-15-methylPGE₂, 20-ethyl PGE₂, and 8-iso-16-cyclohexyl-tetranor PGE₂.

In another additional embodiment, the prostaglandin pathway agonistcomprises PGE₂, or a PGE₂ analogue or derivative thereof.

In another embodiment, the prostaglandin pathway agonist comprises16,16-dimethyl PGE₂.

In another particular embodiment, the glucocorticoid is selected fromthe group consisting of medrysone, alclometasone, alclometasonedipropionate, amcinonide, beclometasone, beclomethasone dipropionate,betamethasone, betamethasone benzoate, betamethasone valerate,budesonide, ciclesonide, clobetasol, clobetasol butyrate, clobetasolpropionate, clobetasone, clocortolone, cloprednol, cortisol, cortisone,cortivazol, deflazacort, desonide, desoximetasone, desoxycortone,desoxymethasone, dexamethasone, diflorasone, diflorasone diacetate,diflucortolone, diflucortolone valerate, difluorocortolone,difluprednate, fluclorolone, fluclorolone acetonide, fludroxycortide,flumetasone, flumethasone, flumethasone pivalate, flunisolide,flunisolide hemihydrate, fluocinolone, fluocinolone acetonide,fluocinonide, fluocortin, fluocoritin butyl, fluocortolone,fluorocortisone, fluorometholone, fluperolone, fluprednidene,fluprednidene acetate, fluprednisolone, fluticasone, fluticasonepropionate, formocortal, halcinonide, halometasone, hydrocortisone,hydrocortisone acetate, hydrocortisone aceponate, hydrocortisonebuteprate, hydrocortisone butyrate, loteprednol, meprednisone,6a-methylprednisolone, methylprednisolone, methylprednisolone acetate,methylprednisolone aceponate, mometasone, mometasone furoate, mometasonefuroate monohydrate, paramethasone, prednicarbate, prednisolone,prednisone, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide and ulobetasol.

In another certain embodiment, the hematopoietic stem or progenitorcells have been contacted for a time of at least about one hour with atleast one of (i) one or more prostaglandin pathway agonists or (ii) oneor more glucocorticoids.

In another further embodiment, the hematopoietic stem or progenitorcells have been contacted for a time of about two hours to abouttwenty-four hours with at least one of (i) one or more prostaglandinpathway agonists and (ii) one or more glucocorticoids.

In another additional embodiment, the hematopoietic stem or progenitorcells have been contacted for a time of about two hours to about sixhours with at least one of (i) one or more prostaglandin pathwayagonists and (ii) one or more glucocorticoids.

In a particular embodiment, the hematopoietic stem or progenitor cellshave been contacted for a time of about four hours with at least one of(i) one or more prostaglandin pathway agonists and (ii) one or moreglucocorticoids.

In a certain embodiment, the population of cells comprises less thanabout 0.10, 0.50, 1.0, 3, 5, 10, 15, 20, or 30% CD34⁺ cells.

In a further embodiment, the population of cells comprises at leastabout 0.01% and no more than about 50% of CD34⁺ cells.

In another embodiment, the population of cells comprises at least about1% CD34⁺ cells.

In an additional embodiment, the population of cells comprises at leastabout 3% CD34⁺ cells.

In a particular embodiment, the population of cells comprises at leastabout 5% CD34⁺ cells.

In another particular embodiment, the population of cells comprises atleast about 10% CD34⁺ cells.

In yet another particular embodiment, the population of cells comprisesat least about 20% CD34⁺ cells.

In still yet another particular embodiment, the population of cellscomprises at least about 30% CD34⁺ cells.

In a certain embodiment, the population of cells comprises at leastabout 40% CD34⁺ cells.

In another certain embodiment, the population of cells comprises atleast about 50% CD34⁺ cells.

In yet another certain embodiment, the population of cells comprises atleast about 60% CD34⁺ cells.

In still yet another certain embodiment, the population of cellscomprises at least about 70% CD34⁺ cells.

In a further embodiment, the population of cells comprises at leastabout 80% CD34⁺ cells.

In another further embodiment, the population of cells comprises atleast about 90% CD34⁺ cells.

In yet another further embodiment, the population of cells comprises atleast about 95% CD34⁺ cells.

In an additional embodiment, the population of cells is not expanded exvivo.

In a certain embodiment, the composition is generated at a point-of-careand is administered into a patient without culturing the population ofcells.

In a further embodiment, the composition is washed and is substantiallyfree of the one or more agents.

In another embodiment, the population of cells is obtained from bonemarrow, fetal liver, fetal blood, placenta, placental blood, umbilicalcord blood, or mobilized peripheral blood.

In one embodiment, the present invention contemplates, in part, a methodof preparing a human hematopoietic stem or progenitor cell comprisingcontacting the hematopoietic stem or progenitor cell ex vivo with one ormore agents that increase CXCR4 gene expression in the cells; whereinthe CXCR4 gene expression is increased at least about 30 fold in thecontacted hematopoietic stem or progenitor cell compared tonon-contacted hematopoietic stem or progenitor cells.

In a particular embodiment, the one or more agents comprises (i) one ormore prostaglandin pathway agonists; and (ii) one or moreglucocorticoids.

In a certain particular embodiment, the prostaglandin pathway agonistcomprises a compound that selectively binds the PGE₂ EP₂ or PGE₂ EP₄receptor.

In a further particular embodiment, the prostaglandin pathway agonist isselected from the group consisting of PGE₂, dmPGE₂, 15(S)-15-methylPGE₂, 20-ethyl PGE₂, and 8-iso-16-cyclohexyl-tetranor PGE₂.

In another particular embodiment, the prostaglandin pathway agonistcomprises PGE₂, or a PGE₂ analogue or derivative.

In an additional particular embodiment, the prostaglandin pathwayagonist comprises 16,16-dimethyl PGE₂.

In a certain embodiment, the glucocorticoid is selected from the groupconsisting of medrysone, alclometasone, alclometasone dipropionate,amcinonide, beclometasone, beclomethasone dipropionate, betamethasone,betamethasone benzoate, betamethasone valerate, budesonide, ciclesonide,clobetasol, clobetasol butyrate, clobetasol propionate, clobetasone,clocortolone, cloprednol, cortisol, cortisone, cortivazol, deflazacort,desonide, desoximetasone, desoxycortone, desoxymethasone, dexamethasone,diflorasone, diflorasone diacetate, diflucortolone, diflucortolonevalerate, difluorocortolone, difluprednate, fluclorolone, flucloroloneacetonide, fludroxycortide, flumetasone, flumethasone, flumethasonepivalate, flunisolide, flunisolide hemihydrate, fluocinolone,fluocinolone acetonide, fluocinonide, fluocortin, fluocoritin butyl,fluocortolone, fluorocortisone, fluorometholone, fluperolone,fluprednidene, fluprednidene acetate, fluprednisolone, fluticasone,fluticasone propionate, formocortal, halcinonide, halometasone,hydrocortisone, hydrocortisone acetate, hydrocortisone aceponate,hydrocortisone buteprate, hydrocortisone butyrate, loteprednol,meprednisone, 6a-methylprednisolone, methylprednisolone,methylprednisolone acetate, methylprednisolone aceponate, mometasone,mometasone furoate, mometasone furoate monohydrate, paramethasone,prednicarbate, prednisolone, prednisone, prednylidene, rimexolone,tixocortol, triamcinolone, triamcinolone acetonide and ulobetasol.

In a further certain embodiment, the glucocorticoid is medrysone.

In an additional certain embodiment, the stem or progenitor cells havebeen contacted with at least one agent for a time of at least about onehour.

In another certain embodiment, the hematopoietic stem or progenitor cellhas been contacted with at least one agent for a time of about one hourto about twenty-four hours.

In another certain embodiment, the stem or progenitor cells have beencontacted with at least one agent for a time of about one hour to aboutsix hours.

In a particular certain embodiment, the stem or progenitor cells havebeen contacted with at least one agent for a time of about two hours toabout six hours.

In a further embodiment, the stem or progenitor cells have beencontacted with at least one agent for a time of about two hours to aboutfour hours.

In an addition further embodiment, the cell is obtained from bonemarrow, umbilical cord blood, mobilized peripheral blood, Wharton'sjelly, placenta, or fetal blood.

In one embodiment, the present invention contemplates, in part, a methodof preparing a therapeutic composition comprising contactinghematopoietic stem or progenitor cells ex vivo with (i) one or moreprostaglandin pathway agonists and (ii) one or more glucocorticoids;wherein the CXCR4 gene expression is increased at least about 30 fold inthe contacted hematopoietic stem or progenitor cells compared tonon-contacted hematopoietic stem or progenitor cells.

In a particular embodiment, gene expression of CXCR4 is increased by atleast about 40 fold in the contacted hematopoietic stem or progenitorcells compared to non-contacted hematopoietic stem or progenitor cells.

In a certain particular embodiment, the prostaglandin pathway agonistcomprises a compound that selectively binds the PGE₂ EP₂ or PGE₂ EP₄receptor.

In a further particular embodiment, the prostaglandin pathway agonist isselected from the group consisting of PGE₂, dmPGE₂, 15(S)-15-methylPGE₂, 20-ethyl PGE₂, and 8-iso-16-cyclohexyl-tetranor PGE₂.

In another particular embodiment, the prostaglandin pathway agonistcomprises PGE₂, or a PGE₂ analogue or derivative.

In an additional particular embodiment, the prostaglandin pathwayagonist comprises 16,16-dimethyl PGE₂.

In a certain embodiment, the glucocorticoid is selected from the groupconsisting of medrysone, alclometasone, alclometasone dipropionate,amcinonide, beclometasone, beclomethasone dipropionate, betamethasone,betamethasone benzoate, betamethasone valerate, budesonide, ciclesonide,clobetasol, clobetasol butyrate, clobetasol propionate, clobetasone,clocortolone, cloprednol, cortisol, cortisone, cortivazol, deflazacort,desonide, desoximetasone, desoxycortone, desoxymethasone, dexamethasone,diflorasone, diflorasone diacetate, diflucortolone, diflucortolonevalerate, difluorocortolone, difluprednate, fluclorolone, flucloroloneacetonide, fludroxycortide, flumetasone, flumethasone, flumethasonepivalate, flunisolide, flunisolide hemihydrate, fluocinolone,fluocinolone acetonide, fluocinonide, fluocortin, fluocoritin butyl,fluocortolone, fluorocortisone, fluorometholone, fluperolone,fluprednidene, fluprednidene acetate, fluprednisolone, fluticasone,fluticasone propionate, formocortal, halcinonide, halometasone,hydrocortisone, hydrocortisone acetate, hydrocortisone aceponate,hydrocortisone buteprate, hydrocortisone butyrate, loteprednol,meprednisone, 6a-methylprednisolone, methylprednisolone,methylprednisolone acetate, methylprednisolone aceponate, mometasone,mometasone furoate, mometasone furoate monohydrate, paramethasone,prednicarbate, prednisolone, prednisone, prednylidene, rimexolone,tixocortol, triamcinolone, triamcinolone acetonide and ulobetasol.

In a further certain embodiment, the glucocorticoid is medrysone.

In a particular embodiment, the hematopoietic stem or progenitor cellshave been contacted with (i) one or more prostaglandin pathway agonistsand (ii) one or more glucocorticoids for a time of at least about onehour.

In a further particular embodiment, the hematopoietic stem or progenitorcells have been contacted with (i) one or more prostaglandin pathwayagonists and (ii) one or more glucocorticoids for a time of about twohours to about twenty-four hours.

In a further particular embodiment, the hematopoietic stem or progenitorcells have been contacted with (i) one or more prostaglandin pathwayagonists and (ii) one or more glucocorticoids for a time of about twohours to about twelve hours.

In a further particular embodiment, the hematopoietic stem or progenitorcells have been contacted with (i) one or more prostaglandin pathwayagonists and (ii) one or more glucocorticoids for a time of about twohours to about six hours.

In an additional particular embodiment, the hematopoietic stem orprogenitor cells have been contacted with (i) one or more prostaglandinpathway agonists and (ii) one or more glucocorticoids for a time ofabout two hours to about four hours.

In another particular embodiment, the hematopoietic stem or progenitorcells have been contacted with (i) one or more prostaglandin pathwayagonists and (ii) one or more glucocorticoids for a time of about fourhours.

In a certain embodiment, the population of cells comprises less thanabout 0.10, 0.50, 1.0, 3, 5, 10, 15, 20, or 30% CD34⁺ cells.

In a further embodiment, the population of cells comprises at leastabout 0.01% and no more than about 50% of CD34⁺ cells.

In another embodiment, the population of cells comprises at least about1% CD34⁺ cells.

In an additional embodiment, the population of cells comprises at leastabout 3% CD34⁺ cells.

In a particular embodiment, the population of cells comprises at leastabout 5% CD34⁺ cells.

In another particular embodiment, the population of cells comprises atleast about 10% CD34⁺ cells.

In yet another particular embodiment, the population of cells comprisesat least about 20% CD34⁺ cells.

In still yet another particular embodiment, the population of cellscomprises at least about 30% CD34⁺ cells.

In a certain embodiment, the population of cells comprises at leastabout 40% CD34⁺ cells.

In another certain embodiment, the population of cells comprises atleast about 50% CD34⁺ cells.

In yet another certain embodiment, the population of cells comprises atleast about 60% CD34⁺ cells.

In still yet another certain embodiment, the population of cellscomprises at least about 70% CD34⁺ cells.

In a further embodiment, the population of cells comprises at leastabout 80% CD34⁺ cells.

In another further embodiment, the population of cells comprises atleast about 90% CD34⁺ cells.

In yet another further embodiment, the population of cells comprises atleast about 95% CD34⁺ cells.

In an additional embodiment, the population of cells is not expanded exvivo.

In a certain embodiment, the composition is generated at a point-of-careand is administered into a patient without culturing the population ofcells.

In a further embodiment, the composition is washed and is substantiallyfree of the one or more agents.

In another embodiment, the population of cells is obtained from bonemarrow, fetal liver, fetal blood, placenta, placental blood, umbilicalcord blood, or mobilized peripheral blood.

In various embodiments, the present invention contemplates, in part, amethod of treating a subject in need of cell therapy comprisingadministering to the subject human hematopoietic stem or progenitorcells wherein the hematopoietic stem or progenitor cells have beencontacted ex vivo one or more agents that increase CXCR4 expression inthe human hematopoietic stem or progenitor cells and gene expression ofCXCR4 is increased at least about 30 fold in the contacted hematopoieticstem or progenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In a particular embodiment, the subject has acute myelogenous leukemia(AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia(CML), chronic lymphocytic leukemia (CLL), juvenile myelomonocyticleukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma,severe aplastic anemia, Fanconi's anemia, paroxysmal nocturnalhemoglobinuria (PNH), pure red cell aplasia,amegakaryocytosis/congenital thrombocytopenia, severe combinedimmunodeficiency syndrome (SCID), Wiskott-Aldrich syndrome,beta-thalassemia major, sickle cell disease, Hurler's syndrome,adrenoleukodystrophy, metachromatic leukodystrophy, myelodysplasia,refractory anemia, chronic myelomonocytic leukemia, agnogenic myeloidmetaplasia, familial erythrophagocytic lymphohistiocytosis, solidtumors, chronic granulomatous disease, mucopolysaccharidoses, or DiamondBlackfan.

In a certain embodiment, the subject has breast cancer, ovarian cancer,brain cancer, prostate cancer, lung cancer, colon cancer, skin cancer,liver cancer, pancreatic cancer, or sarcoma.

In another embodiment, the subject has received bone marrow ablative ornon-myeolablative chemotherapy or radiation therapy.

In a further embodiment, the subject is a bone marrow donor.

In one embodiment, the subject has an ischemic tissue or a tissuedamaged by ischemia.

In a particular embodiment, the subject has at least one symptomassociated with an ischemic tissue or a tissue damaged by ischemia.

In various embodiments, the ischemia is associated with acute coronarysyndrome, acute lung injury (ALI), acute myocardial infarction (AMI),acute respiratory distress syndrome (ARDS), arterial occlusive disease,arteriosclerosis, articular cartilage defect, aseptic systemicinflammation, atherosclerotic cardiovascular disease, autoimmunedisease, bone fracture, bone fracture, brain edema, brain hypoperfusion,Buerger's disease, burns, cancer, cardiovascular disease, cartilagedamage, cerebral infarct, cerebral ischemia, cerebral stroke,cerebrovascular disease, chemotherapy-induced neuropathy, chronicinfection, chronic mesenteric ischemia, claudication, congestive heartfailure, connective tissue damage, contusion, coronary artery disease(CAD), critical limb ischemia (CLI), Crohn's disease, deep veinthrombosis, deep wound, delayed ulcer healing, delayed wound-healing,diabetes (type I and type II), diabetic neuropathy, diabetes inducedischemia, disseminated intravascular coagulation (DIC), embolic brainischemia, graft-versus-host disease, hereditary hemorrhagictelengiectasiaischemic vascular disease, hyperoxic injury, hypoxia,inflammation, inflammatory bowel disease, inflammatory disease, injuredtendons, intermittent claudication, intestinal ischemia, ischemia,ischemic brain disease, ischemic heart disease, ischemic peripheralvascular disease, ischemic placenta, ischemic renal disease, ischemicvascular disease, ischemic-reperfusion injury, laceration, left maincoronary artery disease, limb ischemia, lower extremity ischemia,myocardial infarction, myocardial ischemia, organ ischemia,osteoarthritis, osteoporosis, osteosarcoma, Parkinson's disease,peripheral arterial disease (PAD), peripheral artery disease, peripheralischemia, peripheral neuropathy, peripheral vascular disease,pre-cancer, pulmonary edema, pulmonary embolism, remodeling disorder,renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers,solid organ transplantation, spinal cord injury, stroke,subchondral-bone cyst, thrombosis, thrombotic brain ischemia, tissueischemia, transient ischemic attack (TIA), traumatic brain injury,ulcerative colitis, vascular disease of the kidney, vascularinflammatory conditions, von Hippel-Lindau syndrome, and wounds totissues or organs.

In one embodiment, the present invention contemplates, in part, a methodof increasing hematopoietic stem and progenitor cell homing and/orengraftment in a subject comprising administering to the subject acomposition comprising a population of cells comprising humanhematopoietic stem or progenitor cells wherein the hematopoietic stem orprogenitor cells have been contacted ex vivo with one or more agentsthat increase CXCR4 gene expression in the cells; and gene expression ofCXCR4 is increased at least about 30 fold in the contacted hematopoieticstem or progenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In a particular embodiment, the one or more agents comprises (i) one ormore prostaglandin pathway agonists; and (ii) one or moreglucocorticoids.

In a certain particular embodiment, the prostaglandin pathway agonistcomprises a compound that selectively binds the PGE₂ EP₂ or PGE₂ EP₄receptor.

In a further particular embodiment, the prostaglandin pathway agonist isselected from the group consisting of PGE₂, dmPGE₂, 15(S)-15-methylPGE₂, 20-ethyl PGE₂, and 8-iso-16-cyclohexyl-tetranor PGE₂.

In another particular embodiment, the prostaglandin pathway agonistcomprises PGE₂, or a PGE₂ analogue or derivative.

In an additional particular embodiment, the prostaglandin pathwayagonist comprises 16,16-dimethyl PGE₂.

In a certain embodiment, the glucocorticoid is selected from the groupconsisting of medrysone, alclometasone, alclometasone dipropionate,amcinonide, beclometasone, beclomethasone dipropionate, betamethasone,betamethasone benzoate, betamethasone valerate, budesonide, ciclesonide,clobetasol, clobetasol butyrate, clobetasol propionate, clobetasone,clocortolone, cloprednol, cortisol, cortisone, cortivazol, deflazacort,desonide, desoximetasone, desoxycortone, desoxymethasone, dexamethasone,diflorasone, diflorasone diacetate, diflucortolone, diflucortolonevalerate, difluorocortolone, difluprednate, fluclorolone, flucloroloneacetonide, fludroxycortide, flumetasone, flumethasone, flumethasonepivalate, flunisolide, flunisolide hemihydrate, fluocinolone,fluocinolone acetonide, fluocinonide, fluocortin, fluocoritin butyl,fluocortolone, fluorocortisone, fluorometholone, fluperolone,fluprednidene, fluprednidene acetate, fluprednisolone, fluticasone,fluticasone propionate, formocortal, halcinonide, halometasone,hydrocortisone, hydrocortisone acetate, hydrocortisone aceponate,hydrocortisone buteprate, hydrocortisone butyrate, loteprednol,meprednisone, 6a-methylprednisolone, methylprednisolone,methylprednisolone acetate, methylprednisolone aceponate, mometasone,mometasone furoate, mometasone furoate monohydrate, paramethasone,prednicarbate, prednisolone, prednisone, prednylidene, rimexolone,tixocortol, triamcinolone, triamcinolone acetonide and ulobetasol.

In a further certain embodiment, the glucocorticoid is medrysone.

In an additional certain embodiment, the stem or progenitor cells havebeen contacted with at least one agent for a time of at least about onehour.

In another certain embodiment, the hematopoietic stem or progenitor cellhas been contacted with at least one agent for a time of about one hourto about twenty-four hours.

In another certain embodiment, the stem or progenitor cells have beencontacted with at least one agent for a time of about one hour to aboutsix hours.

In a particular certain embodiment, the stem or progenitor cells havebeen contacted with at least one agent for a time of about two hours toabout six hours.

In a further embodiment, the stem or progenitor cells have beencontacted with at least one agent for a time of about two hours to aboutfour hours.

In an addition further embodiment, the cell is obtained from bonemarrow, umbilical cord blood, mobilized peripheral blood, Wharton'sjelly, placenta, or fetal blood.

In one embodiment, the present invention contemplates, a method ofincreasing hematopoietic stem and progenitor cell homing and/orengraftment in a subject comprising administering to the subject acomposition comprising a population of cells comprising humanhematopoietic stem or progenitor cells wherein the hematopoietic stem orprogenitor cells have been contacted ex vivo with (i) one or moreprostaglandin pathway agonists and (ii) one or more glucocorticoids; andgene expression of CXCR4 is increased at least about 30 fold in thecontacted hematopoietic stem or progenitor cells compared tonon-contacted hematopoietic stem or progenitor cells.

In a particular embodiment, gene expression of CXCR4 is increased by atleast about 40 fold in the contacted hematopoietic stem or progenitorcells compared to non-contacted hematopoietic stem or progenitor cells.

In a certain particular embodiment, the prostaglandin pathway agonistcomprises a compound that selectively binds the PGE₂ EP₂ or PGE₂ EP₄receptor.

In a further particular embodiment, the prostaglandin pathway agonist isselected from the group consisting of PGE₂, dmPGE₂, 15(S)-15-methylPGE₂, 20-ethyl PGE₂, and 8-iso-16-cyclohexyl-tetranor PGE₂.

In another particular embodiment, the prostaglandin pathway agonistcomprises PGE₂, or a PGE₂ analogue or derivative.

In an additional particular embodiment, the prostaglandin pathwayagonist comprises 16,16-dimethyl PGE₂.

In a certain embodiment, the glucocorticoid is selected from the groupconsisting of medrysone, alclometasone, alclometasone dipropionate,amcinonide, beclometasone, beclomethasone dipropionate, betamethasone,betamethasone benzoate, betamethasone valerate, budesonide, ciclesonide,clobetasol, clobetasol butyrate, clobetasol propionate, clobetasone,clocortolone, cloprednol, cortisol, cortisone, cortivazol, deflazacort,desonide, desoximetasone, desoxycortone, desoxymethasone, dexamethasone,diflorasone, diflorasone diacetate, diflucortolone, diflucortolonevalerate, difluorocortolone, difluprednate, fluclorolone, flucloroloneacetonide, fludroxycortide, flumetasone, flumethasone, flumethasonepivalate, flunisolide, flunisolide hemihydrate, fluocinolone,fluocinolone acetonide, fluocinonide, fluocortin, fluocoritin butyl,fluocortolone, fluorocortisone, fluorometholone, fluperolone,fluprednidene, fluprednidene acetate, fluprednisolone, fluticasone,fluticasone propionate, formocortal, halcinonide, halometasone,hydrocortisone, hydrocortisone acetate, hydrocortisone aceponate,hydrocortisone buteprate, hydrocortisone butyrate, loteprednol,meprednisone, 6a-methylprednisolone, methylprednisolone,methylprednisolone acetate, methylprednisolone aceponate, mometasone,mometasone furoate, mometasone furoate monohydrate, paramethasone,prednicarbate, prednisolone, prednisone, prednylidene, rimexolone,tixocortol, triamcinolone, triamcinolone acetonide and ulobetasol.

In a further certain embodiment, the glucocorticoid is medrysone.

In a particular embodiment, the hematopoietic stem or progenitor cellshave been contacted with (i) one or more prostaglandin pathway agonistsand (ii) one or more glucocorticoids for a time of at least about onehour.

In a further particular embodiment, the hematopoietic stem or progenitorcells have been contacted with (i) one or more prostaglandin pathwayagonists and (ii) one or more glucocorticoids for a time of about twohours to about six hours.

In an additional particular embodiment, the hematopoietic stem orprogenitor cells have been contacted with (i) one or more prostaglandinpathway agonists and (ii) one or more glucocorticoids for a time ofabout two hours to about four hours.

In another particular embodiment, the hematopoietic stem or progenitorcells have been contacted with (i) one or more prostaglandin pathwayagonists and (ii) one or more glucocorticoids for a time of about fourhours.

In a certain embodiment, the population of cells comprises less thanabout 0.10, 0.50, 1.0, 3, 5, 10, 15, 20, or 30% CD34⁺ cells.

In a further embodiment, the population of cells comprises at leastabout 0.01% and no more than about 50% of CD34⁺ cells.

In another embodiment, the population of cells comprises at least about1% CD34⁺ cells.

In an additional embodiment, the population of cells comprises at leastabout 3% CD34⁺ cells.

In a particular embodiment, the population of cells comprises at leastabout 5% CD34⁺ cells.

In another particular embodiment, the population of cells comprises atleast about 10% CD34⁺ cells.

In yet another particular embodiment, the population of cells comprisesat least about 20% CD34⁺ cells.

In still yet another particular embodiment, the population of cellscomprises at least about 30% CD34⁺ cells.

In a certain embodiment, the population of cells comprises at leastabout 40% CD34⁺ cells.

In another certain embodiment, the population of cells comprises atleast about 50% CD34⁺ cells.

In yet another certain embodiment, the population of cells comprises atleast about 60% CD34⁺ cells.

In still yet another certain embodiment, the population of cellscomprises at least about 70% CD34⁺ cells.

In a further embodiment, the population of cells comprises at leastabout 80% CD34⁺ cells.

In another further embodiment, the population of cells comprises atleast about 90% CD34⁺ cells.

In yet another further embodiment, the population of cells comprises atleast about 95% CD34⁺ cells.

In an additional embodiment, the population of cells is not expanded exvivo.

In a certain embodiment, the composition is generated at a point-of-careand is administered into a patient without culturing the population ofcells.

In a further embodiment, the composition is washed and is substantiallyfree of the one or more agents.

In another embodiment, the population of cells is obtained from bonemarrow, fetal liver, fetal blood, placenta, placental blood, umbilicalcord blood, or mobilized peripheral blood.

In various preceding embodiments, the subject has acute myelogenousleukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenousleukemia (CML), chronic lymphocytic leukemia (CLL), juvenilemyelomonocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma,multiple myeloma, severe aplastic anemia, Fanconi's anemia, paroxysmalnocturnal hemoglobinuria (PNH), pure red cell aplasia,amegakaryocytosis/congenital thrombocytopenia, severe combinedimmunodeficiency syndrome (SCID), Wiskott-Aldrich syndrome,beta-thalassemia major, sickle cell disease, Hurler's syndrome,adrenoleukodystrophy, metachromatic leukodystrophy, myelodysplasia,refractory anemia, chronic myelomonocytic leukemia, agnogenic myeloidmetaplasia, familial erythrophagocytic lymphohistiocytosis, or solidtumors.

In several preceding embodiments, the subject has breast cancer, ovariancancer, brain cancer, prostate cancer, lung cancer, colon cancer, skincancer, liver cancer, pancreatic cancer, or sarcoma.

In any of the preceding embodiments, the subject has received bonemarrow ablative or non-myeolablative chemotherapy or radiation therapy.

In particular preceding embodiments, the subject is a bone marrow donor.

In certain preceding embodiments, the population of cells is autogeneicto the subject.

In several preceding embodiments, the population of cells is mobilizedfrom the peripheral blood or bone marrow of the subject.

In various preceding embodiments, the population of cells is allogeneicto the subject.

In one embodiment, the present invention contemplates, in part, a methodof increasing hematopoietic stem and progenitor cell reconstitution in asubject comprising administering to the subject a composition comprisinga population of cells comprising human hematopoietic stem or progenitorcells wherein the hematopoietic stem or progenitor cell has beencontacted ex vivo with one or more agents that increase CXCR4 geneexpression in the cells; and gene expression of CXCR4 is increased atleast about 30 fold in the contacted hematopoietic stem or progenitorcell compared to non-contacted hematopoietic stem or progenitor cells.

In a particular embodiment, the one or more agents comprises (i) one ormore prostaglandin pathway agonists; and (ii) one or moreglucocorticoids.

In a certain particular embodiment, the prostaglandin pathway agonistcomprises a compound that selectively binds the PGE₂ EP₂ or PGE₂ EP₄receptor.

In a further particular embodiment, the prostaglandin pathway agonist isselected from the group consisting of PGE₂, dmPGE₂, 15(S)-15-methylPGE₂, 20-ethyl PGE₂, and 8-iso-16-cyclohexyl-tetranor PGE₂.

In another particular embodiment, the prostaglandin pathway agonistcomprises PGE₂, or a PGE₂ analogue or derivative.

In an additional particular embodiment, the prostaglandin pathwayagonist comprises 16,16-dimethyl PGE₂.

In a certain embodiment, the glucocorticoid is selected from the groupconsisting of medrysone, alclometasone, alclometasone dipropionate,amcinonide, beclometasone, beclomethasone dipropionate, betamethasone,betamethasone benzoate, betamethasone valerate, budesonide, ciclesonide,clobetasol, clobetasol butyrate, clobetasol propionate, clobetasone,clocortolone, cloprednol, cortisol, cortisone, cortivazol, deflazacort,desonide, desoximetasone, desoxycortone, desoxymethasone, dexamethasone,diflorasone, diflorasone diacetate, diflucortolone, diflucortolonevalerate, difluorocortolone, difluprednate, fluclorolone, flucloroloneacetonide, fludroxycortide, flumetasone, flumethasone, flumethasonepivalate, flunisolide, flunisolide hemihydrate, fluocinolone,fluocinolone acetonide, fluocinonide, fluocortin, fluocoritin butyl,fluocortolone, fluorocortisone, fluorometholone, fluperolone,fluprednidene, fluprednidene acetate, fluprednisolone, fluticasone,fluticasone propionate, formocortal, halcinonide, halometasone,hydrocortisone, hydrocortisone acetate, hydrocortisone aceponate,hydrocortisone buteprate, hydrocortisone butyrate, loteprednol,meprednisone, 6a-methylprednisolone, methylprednisolone,methylprednisolone acetate, methylprednisolone aceponate, mometasone,mometasone furoate, mometasone furoate monohydrate, paramethasone,prednicarbate, prednisolone, prednisone, prednylidene, rimexolone,tixocortol, triamcinolone, triamcinolone acetonide and ulobetasol.

In a further certain embodiment, the glucocorticoid is medrysone.

In an additional certain embodiment, the stem or progenitor cells havebeen contacted with at least one agent for a time of at least about onehour.

In another certain embodiment, the hematopoietic stem or progenitor cellhas been contacted with at least one agent for a time of about one hourto about twenty-four hours.

In another certain embodiment, the stem or progenitor cells have beencontacted with at least one agent for a time of about one hour to aboutsix hours.

In a particular certain embodiment, the stem or progenitor cells havebeen contacted with at least one agent for a time of about two hours toabout six hours.

In a further embodiment, the stem or progenitor cells have beencontacted with at least one agent for a time of about two hours to aboutfour hours.

In an addition further embodiment, the cell is obtained from bonemarrow, umbilical cord blood, mobilized peripheral blood, Wharton'sjelly, placenta, or fetal blood.

In one embodiment, the present invention contemplates, in part, a methodof increasing hematopoietic stem and progenitor cell reconstitution in asubject comprising administering to the subject a composition comprisinga population of cells comprising human hematopoietic stem or progenitorcells wherein the hematopoietic stem or progenitor cells have beencontacted ex vivo with (i) one or more prostaglandin pathway agonistsand (ii) one or more glucocorticoids; and b) gene expression of CXCR4 isincreased at least about 30 fold in the contacted hematopoietic stem orprogenitor cells compared to non-contacted hematopoietic stem orprogenitor cells.

In a particular embodiment, gene expression of CXCR4 is increased by atleast about 40 fold in the contacted hematopoietic stem or progenitorcells compared to non-contacted hematopoietic stem or progenitor cells.

In a certain particular embodiment, the prostaglandin pathway agonistcomprises a compound that selectively binds the PGE₂ EP₂ or PGE₂ EP₄receptor.

In a further particular embodiment, the prostaglandin pathway agonist isselected from the group consisting of PGE₂, dmPGE₂, 15(S)-15-methylPGE₂, 20-ethyl PGE₂, and 8-iso-16-cyclohexyl-tetranor PGE₂.

In another particular embodiment, the prostaglandin pathway agonistcomprises PGE₂, or a PGE₂ analogue or derivative.

In an additional particular embodiment, the prostaglandin pathwayagonist comprises 16,16-dimethyl PGE₂.

In a certain embodiment, the glucocorticoid is selected from the groupconsisting of medrysone, alclometasone, alclometasone dipropionate,amcinonide, beclometasone, beclomethasone dipropionate, betamethasone,betamethasone benzoate, betamethasone valerate, budesonide, ciclesonide,clobetasol, clobetasol butyrate, clobetasol propionate, clobetasone,clocortolone, cloprednol, cortisol, cortisone, cortivazol, deflazacort,desonide, desoximetasone, desoxycortone, desoxymethasone, dexamethasone,diflorasone, diflorasone diacetate, diflucortolone, diflucortolonevalerate, difluorocortolone, difluprednate, fluclorolone, flucloroloneacetonide, fludroxycortide, flumetasone, flumethasone, flumethasonepivalate, flunisolide, flunisolide hemihydrate, fluocinolone,fluocinolone acetonide, fluocinonide, fluocortin, fluocoritin butyl,fluocortolone, fluorocortisone, fluorometholone, fluperolone,fluprednidene, fluprednidene acetate, fluprednisolone, fluticasone,fluticasone propionate, formocortal, halcinonide, halometasone,hydrocortisone, hydrocortisone acetate, hydrocortisone aceponate,hydrocortisone buteprate, hydrocortisone butyrate, loteprednol,meprednisone, 6a-methylprednisolone, methylprednisolone,methylprednisolone acetate, methylprednisolone aceponate, mometasone,mometasone furoate, mometasone furoate monohydrate, paramethasone,prednicarbate, prednisolone, prednisone, prednylidene, rimexolone,tixocortol, triamcinolone, triamcinolone acetonide and ulobetasol.

In a further certain embodiment, the glucocorticoid is medrysone.

In a particular embodiment, the hematopoietic stem or progenitor cellshave been contacted with (i) one or more prostaglandin pathway agonistsand (ii) one or more glucocorticoids for a time of at least about onehour.

In a further particular embodiment, the hematopoietic stem or progenitorcells have been contacted with (i) one or more prostaglandin pathwayagonists and (ii) one or more glucocorticoids for a time of about twohours to about six hours.

In an additional particular embodiment, the hematopoietic stem orprogenitor cells have been contacted with (i) one or more prostaglandinpathway agonists and (ii) one or more glucocorticoids for a time ofabout two hours to about four hours.

In another particular embodiment, the hematopoietic stem or progenitorcells have been contacted with (i) one or more prostaglandin pathwayagonists and (ii) one or more glucocorticoids for a time of about fourhours.

In a certain embodiment, the population of cells comprises less thanabout 0.10, 0.50, 1.0, 3, 5, 10, 15, 20, or 30% CD34⁺ cells.

In a further embodiment, the population of cells comprises at leastabout 0.01% and no more than about 50% of CD34⁺ cells.

In another embodiment, the population of cells comprises at least about1% CD34⁺ cells.

In an additional embodiment, the population of cells comprises at leastabout 3% CD34⁺ cells.

In a particular embodiment, the population of cells comprises at leastabout 5% CD34⁺ cells.

In another particular embodiment, the population of cells comprises atleast about 10% CD34⁺ cells.

In yet another particular embodiment, the population of cells comprisesat least about 20% CD34⁺ cells.

In still yet another particular embodiment, the population of cellscomprises at least about 30% CD34⁺ cells.

In a certain embodiment, the population of cells comprises at leastabout 40% CD34⁺ cells.

In another certain embodiment, the population of cells comprises atleast about 50% CD34⁺ cells.

In yet another certain embodiment, the population of cells comprises atleast about 60% CD34⁺ cells.

In still yet another certain embodiment, the population of cellscomprises at least about 70% CD34⁺ cells.

In a further embodiment, the population of cells comprises at leastabout 80% CD34⁺ cells.

In another further embodiment, the population of cells comprises atleast about 90% CD34⁺ cells.

In yet another further embodiment, the population of cells comprises atleast about 95% CD34⁺ cells.

In an additional embodiment, the population of cells is not expanded exvivo.

In a certain embodiment, the composition is generated at a point-of-careand is administered into a patient without culturing the population ofcells.

In a further embodiment, the composition is washed and is substantiallyfree of the one or more agents.

In another embodiment, the population of cells is obtained from bonemarrow, fetal liver, fetal blood, placenta, placental blood, umbilicalcord blood, or mobilized peripheral blood.

In various embodiments, the population of cells comprises one or morecord blood units.

In various preceding embodiments, the subject has acute myelogenousleukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenousleukemia (CML), chronic lymphocytic leukemia (CLL), juvenilemyelomonocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma,multiple myeloma, severe aplastic anemia, Fanconi's anemia, paroxysmalnocturnal hemoglobinuria (PNH), pure red cell aplasia,amegakaryocytosis/congenital thrombocytopenia, severe combinedimmunodeficiency syndrome (SCID), Wiskott-Aldrich syndrome,beta-thalassemia major, sickle cell disease, Hurler's syndrome,adrenoleukodystrophy, metachromatic leukodystrophy, myelodysplasia,refractory anemia, chronic myelomonocytic leukemia, agnogenic myeloidmetaplasia, familial erythrophagocytic lymphohistiocytosis, or solidtumors.

In several preceding embodiments, the subject has breast cancer, ovariancancer, brain cancer, prostate cancer, lung cancer, colon cancer, skincancer, liver cancer, pancreatic cancer, or sarcoma.

In any of the preceding embodiments, the subject has received bonemarrow ablative or non-myeolablative chemotherapy or radiation therapy.

In particular preceding embodiments, the subject is a bone marrow donor.

In certain preceding embodiments, the population of cells is autogeneicto the subject.

In several preceding embodiments, the population of cells is mobilizedfrom the peripheral blood or bone marrow of the subject.

In various preceding embodiments, the population of cells is allogeneicto the subject.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the increase in levels of CXCR4 mRNA detected (relative tovehicle treatment) in human cord blood CD34⁺ cells when the cells aretreated with either 10 μM of a single agent or with a combination of 10μM dmPGE₂ and 10 μM of one of five different glucocorticoids.Glucocorticoids act synergistically with dmPGE₂ to increase CXCR4 geneexpression.

FIGS. 2A-2C show the increase in CXCR4 mRNA detected in human cord bloodCD34⁺ cells when the CD34⁺ cells are treated with: PGE₂ alone or acombination of PGE₂ with various glucocorticoids (FIG. 2A);15(S)-15-methyl PGE₂ (mPGE₂) alone or in combination with variousglucocorticoids (FIG. 2B); or 20-ethyl PGE2 (ePGE₂) alone or incombination with various glucocorticoids (FIG. 2C). The datademonstrates that glucocorticoids act synergistically with prostaglandinpathway agonists to increase CXCR4 gene expression in CD34⁺ cells.

FIG. 3 shows the increase in CXCR4 mRNA detected in human CD34⁺ cellsderived from cord blood or mobilized peripheral blood (mPB) when theCD34⁺ cells are treated with either a prostaglandin pathway agonistalone or in combination with a glucocorticoid. CD34⁺ cells respondsimilarly to treatment regardless of source of origin of the CD34⁺cells.

FIGS. 4A-4B show the increase in the number of CD34⁺ cells expressingCXCR4 surface protein (FIG. 4A), depicted as % CXCR4+, and the increasein the amount of CXCR4 surface protein on the CD34⁺ cells (FIG. 4B)measured in Mean Fluorescent Intensity (MFI) after the CD34⁺ cells aretreated with either a prostaglandin pathway agonist alone or incombination with a glucocorticoid.

FIG. 5 shows the kinetic measurement of the increase in CXCR4 mRNAdetected (fold change) and the number of human CD34⁺ cells expressingsurface CXCR4 protein (% Cells CXCR4+) during a 2 hour treatment and foran additional 4 hours post-removal of treatment (media alone) after theCD34⁺ cells are treated with either a prostaglandin pathway agonistalone or in combination with a glucocorticoid.

FIG. 6 shows the kinetic measurement of the increase in CXCR4 mRNAdetected (Fold Change) and the number of human CD34⁺ cells expressingsurface CXCR4 protein (% Cells CXCR4+) during a 4 hour treatment and foran additional 4 hours post removal of treatment (media alone) after theCD34⁺ cells are treated with either a prostaglandin pathway agonistalone or in combination with a glucocorticoid.

FIG. 7 shows the results from a representative SDF-1 transwell migrationassay. The results show the effect of treating CD34⁺ cells with DMSOcontrol, dmPGE₂, or dmPGE₂ and medrysone on the efficiency of cellmigration towards SDF-1. The data demonstrates the enhanced ability ofCD34⁺ cells treated with a combination of a prostaglandin pathwayagonist and a glucocorticoid to migrate to an SDF-1 gradient incomparison to CD34⁺ cells treated with vehicle or a prostaglandinpathway agonist alone. The results indicate that the increase in CXCR4gene expression in cells treated with the combination of a prostaglandinpathway agonist and a glucocorticoid translates to increased functionalability.

FIG. 8 shows the efficiency of cell migration towards SDF-1 of CD34⁺cells treated with dmPGE₂ alone or in combination with variousglucocorticoids. CD34⁺ cells treated with the combination of aprostaglandin pathway agonist and a glucocorticoid show increasedability to migrate towards an SDF-1 gradient as compared to CD34⁺ cellstreated with vehicle or a prostaglandin pathway agonist alone.

FIG. 9 shows the duration of the enhanced migration ability of CD34⁺cells treated with a combination of a prostaglandin pathway agonist anda glucocorticoid. The results demonstrate that the duration of theenhanced migration ability is maintained for at least 4 hours aftertreatment of CD34⁺ cells.

FIG. 10 shows the Neurological Severity Score (mNSS) results from arepresentative middle cerebral artery occlusion model (MCAO) ischemiarat model. The results show the effect of treating HSPCs cells withdmPGE₂ and medrysone on the ability of the cells to reduce neurologicaldeficits in the MCAO stroke model. Neurological deficits are reduced,and neurological function is improved, in rats given HSPCs treated withthe combination of a prostaglandin pathway agonist and a glucocorticoid,as compared to rats given untreated cells or vehicle alone.

FIG. 11 shows the foot-fault assay results from a representative middlecerebral artery occlusion model (MCAO) ischemia rat model. The resultsshow the effect of treating HSPCs with dmPGE₂ and medrysone on theability of the cells to reduce locomotor deficits in the MCAO strokemodel. Locomotor deficit is improved in rats given HSPCs treated withthe combination of a prostaglandin pathway agonist and a glucocorticoid,as compared to rats given untreated cells or vehicle alone.

DETAILED DESCRIPTION

A. Overview

The invention provides human hematopoietic stem and progenitor cellsthat are treated ex vivo to enhance the therapeutic properties of thecells. In particular, the hematopoietic stem and progenitor cells of theinvention have been modified ex vivo by briefly treating the cells withone or more agents that increase gene expression of genes involved inhoming. In one embodiment, hematopoietic stem and progenitor cells ofthe invention have been modified ex vivo by briefly treating the cellswith one or more agents that increase CXCR4 expression. The therapeuticcells of the invention express unexpectedly high levels of CXCR4compared to untreated human hematopoietic stem and progenitor cells. Inparticular embodiments, the pharmacologically enhanced cells of theinvention are characterized by an increase in gene expression of CXCR4of at least about 30 fold compared to untreated cells. In variousembodiments, the therapeutic cells are CD34⁺ cells.

CXCR4 is believed to be associated with increased homing and engraftmentof hematopoietic stem and progenitor cells, and therefore the treatedhematopoietic stem and progenitor cells of the invention have enhancedtherapeutic properties, including for example, increased homing to bonemarrow and ischemia-damaged tissue, as well as enhanced proliferativeand regenerative properties.

In various embodiments, cells of the invention and compositionscontaining such enhanced cells are useful for treating conditions anddisorders where increased numbers of hematopoietic stem and progenitorcells are needed or beneficial, including among other treatments,hematopoietic stem cell transplants and in treating ischemia-damagedtissue. Without wishing to be bound by theory, the present inventioncontemplates, in part, that the increased levels of CXCR4 protein on thesurface of the enhanced hematopoietic stem and progenitor cells of theinvention improve homing of the enhanced cells to the bone marrow and tosites of tissue injury. The enhanced hematopoietic stem and progenitorcells may improve patient outcome during stem cell transplants byincreasing the efficacy of hematopoietic stem and progenitor cells usedin stem cell transplants, including for example, by increasing homingand/or engraftment of treated cells to the bone marrow, and increasingthe ability of treated cells to self-renew and proliferate in vivo afteradministration to a patient.

The enhanced hematopoietic stem and progenitor cells of the inventionmay improve patient outcome when used for treating ischemic tissue orischemia-damaged tissue by, for example, improving vascularization inischemic tissue, improving tissue regeneration at sites of ischemia,decreasing ischemic tissue necrosis or apoptosis, and/or increasing cellsurvival at sites of ischemia.

B. Definitions

The articles “a,” “an,” and “the” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the terms “include” and “comprise” are used synonymously.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length. In oneembodiment, the term “about” or “approximately” refers a range ofquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length ±15%, ±10%, +9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%,±2%, or ±1% about a reference quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length.

The term “ex vivo” refers generally to activities that take placeoutside an organism, such as experimentation or measurements done in oron living tissue in an artificial environment outside the organism,preferably with minimum alteration of the natural conditions. Inparticular embodiments, “ex vivo” procedures involve living cells ortissues taken from an organism and cultured in a laboratory apparatus,usually under sterile conditions, and typically for a few hours or up toabout 24 hours, but including up to 48 or 72 hours, depending on thecircumstances. In certain embodiments, such tissues or cells can becollected and frozen, and later thawed for ex vivo treatment. Tissueculture experiments or procedures lasting longer than a few days usingliving cells or tissue are typically considered to be “in vitro,” thoughin certain embodiments, this term can be used interchangeably with exvivo.

The recitations “ex vivo administration,” “ex vivo treatment,” or “exvivo therapeutic use,” relate generally to medical procedures in whichone or more organs, cells, or tissues are obtained from a living orrecently deceased subject, optionally purified/enriched, exposed to atreatment or procedure (e.g., an ex vivo administration step thatinvolves incubating the cells with a composition or agent of the presentinvention to enhance expansion of particular cells, such ashematopoietic stem or progenitor cells). Cells treated ex vivo may beadministered to the donor or to a different living subject.

Such ex vivo therapeutic applications may also include an optional invivo treatment or procedural step, such as by administering contactedcells of the invention one or more times to the living subject. Bothlocal and systemic administration is contemplated for these embodiments,according to well-known techniques in the art and as described elsewhereherein. The amount of cells administered to a subject will depend on thecharacteristics of that subject, such as general health, age, sex, bodyweight, and tolerance to drugs, as well as the degree, severity, andtype of reaction to the drug and/or cell transplant.

The term “in vivo” refers generally to activities that take place insidean organism, such as cell engraftment, cell homing, self-renewal ofcells, and expansion of cells. In one embodiment, the term “in vivoexpansion” refers to the ability of a cell population to increase innumber in vivo. In particular embodiments, the in vivo expansion includeself-renewal and/or proliferation of stem cells.

By “enhance” or “promote,” or “increase” or “activate” refers generallyto the ability of an agent to produce or cause a greater physiologicalresponse (i.e., downstream effects) in a cell, as compared to theresponse caused by either vehicle or a control molecule/composition,e.g., increased engraftment/engraftment potential of hematopoietic stemand progenitor cells and increased in vivo stem cell expansion. Ameasurable physiological response may include an increase inhematopoietic stem and progenitor cell engraftment, viability, homing,self-renewal, and/or expansion, among others apparent from theunderstanding in the art and the description herein. In one embodiment,the increase can be an increase in gene expression as a result ofincreased signaling through the PGE₂R₂ and/or PGE₂R₄ cell signalingpathways, including, but not limited to an increase in CREBphosphorylation, an increase in CREM expression, and an increase inCXCR4 expression. Increases in hematopoietic stem and progenitor cellengraftment, viability, homing, self-renewal and/or in vivo expansion,can also be ascertained using methods known in the art, such as geneexpression, CFU-C assays, CFU-S assays, CAFC assays, and cell surfaceprotein expression, among others. An “increased” or “enhanced” amount istypically a “statistically significant” amount, and may include anincrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30or more times (e.g., 500, 1000 times) (including all integers anddecimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.)the response produced by vehicle (the absence of an agent) or a controlcomposition.

By “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refersgenerally to the ability of an agent to produce or cause a lesserphysiological response (i.e., downstream effects) in a cell, as comparedto the response caused by either vehicle or a controlmolecule/composition, e.g., decreased apoptosis. In one embodiment, thedecrease can be a decrease in gene expression or a decrease in cellsignaling that normally is associated with a reduction of cellviability. An “decrease” or “reduced” amount is typically a“statistically significant” amount, and may include an decrease that is1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times(e.g., 500, 1000 times) (including all integers and decimal points inbetween and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) the responseproduced by vehicle (the absence of an agent) or a control composition.

By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “nosubstantial change,” or “no substantial decrease” refers generally tothe ability of a agent to produce or cause a comparable physiologicalresponse (i.e., downstream effects) in a cell, as compared to theresponse caused by either vehicle or a control molecule/composition(reference response). A comparable response is one that is notsignificantly different or measurably different from the referenceresponse.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

In certain embodiments, therapeutic cells of the invention comprise aunique or substantially unique gene signature. As used herein, the term“gene expression profile,” “gene expression signature” or “genesignature” refers to the levels of expression of multiple differentgenes measured for the same sample, i.e., a population of cells. A geneexpression signature may be defined so as to identify a group of genes“signature genes” that serves to distinguish the therapeutic cells fromexisting cells in the art and/or control, vehicle, or non-treated cells.

A “signature gene”, as used herein, means any gene in a signature geneset. For example, signature genes include hyaluronan synthase 1 (HAS1),GTP-binding protein GEM (GEM), dual specificity protein phosphatase 4(DUSP4), amphiregulin (AREG), Nuclear receptor related 1 protein(NR4A2), renin (REN), cAMP-responsive element modulator (CREM),collagen, type I, alpha 1 (COL1A1), Fos-related antigen 2 (FOSL2), andCXC chemokine receptor 4 (CXCR4). For clarity, signature genes do notinclude housekeeping genes.

“Gene expression” as used herein refers to the relative levels ofexpression and/or pattern of expression of a gene in a biologicalsample, such as the stem and progenitor cells, or population of cellscomprising stem or progenitor cells. In particular embodiments, the stemor progenitor cells are hematopoietic stem and progenitor cells.

Any methods available in the art for detecting expression of the genescharacterizing the cells comprising the therapeutic composition of theinvention are encompassed herein. As used herein, the term “detectingexpression” means determining the quantity or presence of an RNAtranscript or its expression product of a gene. Methods for detectingexpression of genes, that is, gene expression profiling, include methodsbased on hybridization analysis of polynucleotides, methods based onsequencing of polynucleotides, immunohistochemistry methods, andproteomics-based methods. The methods generally detect expressionproducts (e.g., mRNA) of the genes of interest. In some embodiments,PCR-based methods, such as reverse transcription PCR (RT-PCR) (Weis etal., TIG 8:263-64, 1992), and array-based methods such as microarray(Schena et al., Science 270:467-70, 1995) are used.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

C. Hematopoietic Stem and Progenitor Cells

The invention provides human hematopoietic stem and progenitor cellswherein the stem cells have been contacted ex vivo with one or moreagents capable of increasing the therapeutic properties of the cell. Inone embodiment, human hematopoietic stem and progenitor cells have beencontacted ex vivo with one or more agents that increase CXCR4 geneexpression in the cells. In one preferred embodiment, the geneexpression of CXCR4 is increased in the treated human hematopoietic stemcells at least about 30 fold compared to non-contacted hematopoieticstem and progenitor cells or cells treated with a vehicle control.

Hematopoietic stem cells are multipotent stem cells that give rise toall the blood cell types of an organism, including myeloid (e.g.,monocytes and macrophages, neutrophils, basophils, eosinophils,erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoidlineages (e.g., T-cells, B-cells, NK-cells), and others known in the art(See Fei, R., et al., U.S. Pat. No. 5,635,387; McGlave, et al., U.S.Pat. No. 5,460,964; Simmons, P., et al, U.S. Pat. No. 5,677,136;Tsukamoto, et al., U.S. Pat. No. 5,750,397; Schwartz, et al., U.S. Pat.No. 5,759,793; DiGuisto, et al., U.S. Pat. No. 5,681,599; Tsukamoto, etal., U.S. Pat. No. 5,716,827). Hematopoietic progenitor cells (HSCs)give rise to committed hematopoietic progenitor cells (HPCs) that arecapable of generating the entire repertoire of mature blood cells overthe lifetime of an organism.

As used herein, the term “hematopoietic stem and progenitor cell” or“HSPC” refers to a cell identified by the presence of the antigenicmarker CD34 (CD34⁺) and are therefore characterized as CD34⁺ cells, andpopulations of such cells. In particular embodiments, the term “HSPC”refers to a cell identified by the presence of the antigenic marker CD34(CD34⁺) and the absence of lineage (lin) markers and are thereforecharacterized as CD34⁺/Lin(−) cells, and populations of such cells. Itis recognized that the population of cells comprising CD34⁺ and/orLin(−) cells also includes hematopoietic progenitor cells, and so forthe purposes of this application the term “HSPC” includes hematopoieticprogenitor cells.

“Enhanced hematopoietic stem and progenitor cell” or “enhanced HSPC”refers to a HSPC treated ex vivo with one or more agents that increaseCXCR4 gene expression in the cell at least about 30 fold compared tocontrol, vehicle or untreated cells.

As used herein, a “non-contacted” or an “untreated” cell is a cell thathas not been treated, e.g., cultured, contacted, or incubated with anagent other than a control agent. Cells contacted with DMSO (a controlagent), or contacted with another vehicle are non-contacted cells.

The HSPCs of the invention are identified and are characterized by, agene expression profile indicating high levels of CXCR4 expression. TheHSPCs can also be characterized based upon increased CXCR4 geneexpression and increased cell surface expression of CXCR4 polypeptide.In certain embodiments, the CXCR4 gene expression in the HSPCs of theinvention is increased by at least 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100 fold compared to the expression of CXCR4 innon-contacted cells.

In particular embodiments, CXCR4 gene expression in the HSPCs isincreased by about 30 to about 80 fold compared to untreated HSPCs. Infurther embodiments, CXCR4 gene expression in the HSPCs is increased byabout 40 to about 80 fold, about 50 to about 80 fold, about 60 to about80 fold, or about 50 to about 70 fold, compared to untreated HSPCs.

CXCR4 gene expression or the gene expression signature of the treatedHSPCs of the invention may be determined after the cells are treatedwith one or more agents. For example, HSPCs may be treated ex vivo withone or more agents, washed to remove the agent(s), and the geneexpression analyzed without further incubation of the cells.

Human HSPCs contacted in the methods of the invention and havingenhanced therapeutic properties can also be characterized in multipleand various other ways, such as by increased levels of intracellularcAMP signaling, e.g., CREB phosphorylation, or as determined by abiochemical assay; gene expression signatures indicating upregulation ofgenes implicated in the PGE₂R₂/R4 cell signaling pathway, e.g., CREM,and genes that increase stem and progenitor cell homing and engraftment,e.g., CXCR4, as determined by gene expression assays, e.g., microarrays;no measurable decrease in stem and progenitor cell viability asdetermined by cell viability assays, e.g., 7-aminoactinomycinD (7-AAD)staining; and/or an increased capacity of stem cells to self-renew asdetermined by an in vitro colony forming units (CFU-C) assay, forexample.

1. Determining Gene Expression

“Gene expression” as used herein refers to the relative levels ofexpression and/or pattern of expression of a gene, such as CXCR4, in abiological sample, such as stem and progenitor cells, or a population ofcells comprising stem or progenitor cells, in a therapeutic compositionof the invention. A sample may comprise heterogeneous or homogenouspopulation of cells and the cell populations may be purified or notpurified from the sample. The expression of a gene, such as CXCR4, maybe measured at the level of cDNA, RNA, mRNA, or combinations thereof.

Any methods available in the art for detecting expression of the CXCR4gene are encompassed herein. As used herein, the term “detectingexpression” means determining the quantity or presence of an RNAtranscript or its expression product of a gene. Methods for detectingexpression of genes include methods based on PCR, hybridization analysisof polynucleotides, methods based on sequencing of polynucleotides,immunohistochemistry methods, and proteomics-based methods. The methodsgenerally detect expression products (e.g., mRNA) of the genes ofinterest. In some embodiments, PCR-based methods, such as reversetranscription PCR (RT-PCR) (Weis et al., TIG 8:263-64, 1992), andarray-based methods such as microarray (Schena et al., Science270:467-70, 1995) are used.

General methods for RNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al., ed., Current Protocols in Molecular Biology, John Wiley & Sons,New York 1987-1999. In particular, RNA isolation can be performed usinga purification kit, a buffer set and protease from commercialmanufacturers, such as Qiagen (Valencia, Calif.), according to themanufacturer's instructions. For example, total RNA from cells inculture can be isolated using Qiagen RNeasy mini-columns. Isolated RNAcan be used in hybridization or amplification assays that include, butare not limited to, PCR analyses and probe arrays. One method for thedetection of RNA levels involves contacting the isolated RNA with anucleic acid molecule (probe) that can hybridize to the mRNA encoded bythe gene being detected. The nucleic acid probe can be, for example, afull-length cDNA, or a portion thereof, such as an oligonucleotide of atleast 7, 15, 30, 60, 100, 250, or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to anintrinsic gene of the present invention, or any derivative DNA or RNA.Hybridization of an mRNA with the probe indicates that the intrinsicgene in question is being expressed.

An alternative method for determining the level of gene expression in asample involves the process of nucleic acid amplification, for example,by RT-PCR (U.S. Pat. No. 4,683,202), ligase chain reaction (Barany,Proc. Natl. Acad. Sci. USA 88:189-93, 1991), self sustained sequencereplication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-78,1990), transcriptional amplification system (Kwoh et al., Proc. Natl.Acad. Sci. USA 86:1173-77, 1989), Q-Beta Replicase (Lizardi et al.,Bio/Technology 6:1197, 1988), rolling circle replication (U.S. Pat. No.5,854,033), or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art.

In particular aspects of the invention, gene expression of CXCR4 isassessed by quantitative RT-PCR. Numerous different PCR or QPCRprotocols are known in the art and exemplified herein below and can bedirectly applied or adapted for use using the presently-describedcompositions for the detection and/or quantification of CXCR4.Quantitative PCR (QPCR) (also referred as real-time PCR) is preferredunder some circumstances because it provides not only a quantitativemeasurement, but also reduced time and contamination. In some instances,the availability of full gene expression profiling techniques is limiteddue to requirements for fresh frozen tissue and specialized laboratoryequipment, making the routine use of such technologies difficult in aclinical setting. As used herein, “quantitative PCR (or “real timeQPCR”) refers to the direct monitoring of the progress of PCRamplification as it is occurring without the need for repeated samplingof the reaction products. In quantitative PCR, the reaction products maybe monitored via a signaling mechanism (e.g., fluorescence) as they aregenerated and are tracked after the signal rises above a backgroundlevel but before the reaction reaches a plateau. The number of cyclesrequired to achieve a detectable or “threshold” level of fluorescencevaries directly with the concentration of amplifiable targets at thebeginning of the PCR process, enabling a measure of signal intensity toprovide a measure of the amount of target nucleic acid in a sample inreal time.

“Normalization” may be used to remove sample-to-sample variation. Formicroarray data, the process of normalization aims to remove systematicerrors by balancing the fluorescence intensities of the two labelingdyes. The dye bias can come from various sources including differencesin dye labeling efficiencies, heat and light sensitivities, as well asscanner settings for scanning two channels. Some commonly used methodsfor calculating normalization factor include: (i) global normalizationthat uses all genes on the array, such as by log scale robustmulti-array analysis (RMA); (ii) housekeeping genes normalization thatuses constantly expressed housekeeping/invariant genes; and (iii)internal controls normalization that uses known amount of exogenouscontrol genes added during hybridization (Quackenbush (2002) Nat. Genet.32 (Suppl.), 496-501). In one embodiment, expression of the genesdisclosed herein can be determined by normalizing the expression tocontrol housekeeping gene expression or by performing log scale robustmulti-array analysis (RMA).

2. Gene Expression Profile of Stem or Progenitor Cells

The therapeutic compositions comprise a population of treated stem orprogenitor cells having increased therapeutic properties related to thetreatment of ischemic tissue. Without wishing to be bound to anyparticular theory, treatment of the cells with a prostaglandin pathwayagonist and/or a glucocorticoid imbues the cells with the increasedtherapeutic properties useful for treating ischemic tissue or more ormore symptoms associated with an ischemic tissue. Cells that have theincreased therapeutics properties are characterized by increased CXCR4gene expression and increased cell surface expression of CXCR4polypeptide. In a particular embodiment, the therapeutic compositioncomprises hematopoietic stem or progenitor cells characterized byincreased levels of gene and cell-surface CXCR4 expression.

Stem or progenitor cells, e.g., hematopoietic stem or progenitor cells,treated with a prostaglandin pathway agonist and a glucocorticoid can becharacterized by at least a 40, 45, 50, 55, 60, 65, 70, 75, or 80 foldincrease in CXCR4 gene expression compared to the expression of CXCR4 inuntreated cells.

Cells that have increased therapeutic properties can furthercharacterized by a unique gene expression signature wherein expressionof 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of the signature genes selectedfrom the group consisting of: CXCR4, hyaluronan synthase 1 (HAS1),GTP-binding protein GEM (GEM), dual specificity protein phosphatase 4(DUSP4), amphiregulin (AREG), Nuclear receptor related 1 protein(NR4A2), renin (REN), cAMP-responsive element modulator (CREM),collagen, type I, alpha 1 (COL1A1), and Fos-related antigen 2 (FOSL2) isincreased, compared to untreated cells.

In other particular embodiments, hematopoietic stem or progenitor cellstreated with a prostaglandin pathway agonist and a glucocorticoid have agene expression signature, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreof the signature genes is increased by at least 40, 45, 50, 55, 60, 65,70, 75, or 80 fold compared to untreated cells. In some embodiments, theaverage fold change of all signature genes is at least about 15, 20, 25,30, or 35 fold. In some embodiments, the average fold change of allsignature genes is at least about 25, or 30 fold.

The gene expression or gene expression signature of the treated stem orprogenitor cells may be determined after cells are treated with anagent, or cells may be incubated for some period of time after treatmentbefore determining the gene expression signature of the cells. Forexample, cells may be treated ex vivo with one or more agents, washed toremove the agents, and the gene expression analyzed without furtherincubation of the cells. Alternatively, in some embodiments, cells aretreated with one or more agents, washed to remove the agents from thecell population, and then the cells are incubated ex vivo for someperiod of time prior to analyzing the gene expression signature of thecells.

3. Sources of HSPCs

The HSPCs prepared in the methods of the invention may be obtained fromany suitable source of hematopoietic stem and progenitor cells, and maybe provided, and treated, as a highly purified population of HSPCs (ahomogenous population), or as a composition that comprises from 0.01% toabout 100% of HSPCs (a heterogeneous population). For example, andwithout limitation, HSPCs may be provided in compositions such asunfractionated bone marrow (in which CD34⁺ cells comprise less thanabout 1% of the bone marrow cell population), umbilical cord blood,placental blood, placenta, fetal blood, fetal liver, fetal spleen,Wharton's jelly, or mobilized peripheral blood.

Suitable sources of HSPCs for use in the methods of the inventioninclude, but are not limited to, cells isolated or obtained from anorgan of the body containing cells of hematopoietic origin. By“isolated” is meant material that is removed from its originalenvironment. For example, a cell is isolated if it is separated fromsome or all of the components that normally accompany it in its nativestate. For example, an “isolated population of cells,” an “isolatedsource of cells,” or “isolated HSPCs” and the like, as used herein,refer to in vitro or ex vivo separation of one or more cells from theirnatural cellular environment, and from association with other componentsof the tissue or organ, i.e., it is not significantly associated with invivo substances.

HSPCs can be obtained or isolated from bone marrow of adults, whichincludes femurs, hip, ribs, sternum, and other bones. Bone marrowaspirates containing HSPCs can be obtained or isolated directly from thehip using a needle and syringe. Other sources of HSPCs include umbilicalcord blood, placental blood, mobilized peripheral blood, Wharton'sjelly, placenta, fetal blood, fetal liver, or fetal spleen. Inparticular embodiments, harvesting a sufficient quantity of HSPCs foruse in therapeutic applications may require mobilizing the stem andprogenitor cells in the donor.

“Hematopoietic stem cell mobilization” refers to the release of stemcells from the bone marrow into the peripheral blood circulation for thepurpose of leukapheresis, prior to stem cell transplantation. Byincreasing the number of stem cells harvested from the donor, the numberof stem cells available for therapeutic applications can besignificantly improved. Hematopoietic growth factors, e.g., granulocytecolony stimulating factor (G-CSF) or chemotherapeutic agents often areused to stimulate the mobilization. Commercial stem cell mobilizationdrugs exist and can be used in combination with G-CSF to mobilizesufficient quantities of hematopoietic stem and progenitor cells fortransplantation into a subject. For example, G-CSF and Mozobil™ (GenzymeCorporation) can be administered to a donor in order to harvest asufficient number of hematopoietic cells for transplantation. Othermethods of mobilizing hematopoietic stem and progenitor cells would beapparent to one having skill in the art.

In particular embodiments, HSPCs are obtained from umbilical cord blood.Cord blood can be harvested according to techniques known in the art(see, e.g., U.S. Pat. Nos. 7,147,626 and 7,131,958, herein incorporatedby reference for such methodologies).

In one embodiment, HSPCs can be obtained from pluripotent stem cellsources, e.g., induced pluripotent stem cells (iPSCs) and embryonic stemcells (ESCs). As used herein, the term “induced pluripotent stem cell”or “iPSC” refers to a non-pluripotent cell that has been reprogrammed toa pluripotent state. Once the cells of a subject have been reprogrammedto a pluripotent state, the cells can then be programmed to a desiredcell type, such as a hematopoietic stem or progenitor cell. As usedherein, the term “reprogramming” refers to a method of increasing thepotency of a cell to a less differentiated state. As used herein, theterm “programming” refers to a method of decreasing the potency of acell or differentiating the cell to a more differentiated state.

4. Therapeutic Cellular Compositions

The invention also provides therapeutic compositions comprising theenhanced HSPCs described herein. In particular, the therapeuticcompositions of the invention comprise a population of cells comprisingHSPCs wherein the HSPCs have been contacted ex vivo with one or moreagents capable of increasing CXCR4 gene expression in the HSPCs, andwherein the gene expression of CXCR4 is increased in the HSPCs by atleast about 30 fold relative to non-contacted HSPCs. In one embodiment,the therapeutic compositions of the invention comprise a population ofcells comprising HSPCs treated ex vivo with a prostaglandin pathwayagonist and a glucocorticoid. In certain embodiments, the therapeuticcomposition comprising the enhanced HSPCs is whole bone marrow,umbilical cord blood, or mobilized peripheral blood.

In particular embodiments, the therapeutic composition comprises apopulation of cells, wherein the population of cells is about 95% toabout 100% HSPCs. The invention contemplates, in part, that usingtherapeutic compositions of highly purified HSPCs, e.g., a compositioncomprising a population of cells wherein the cells comprise about 95%HSPCs, may improve the efficiency of stem cell therapies. Currentlypracticed methods of transplantations typically use unfractionatedmixtures of cells where HSPCs comprise less than 1% of the total cellpopulation.

In some embodiments, the therapeutic composition comprises a populationof cells, wherein the population of cells comprises less than about0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% HSPCs. The populationof cells in some embodiments comprises less than about 0.1%, 0.5%, 1%,2%, 5%, 10%, 15%, 20%, 25%, or 30% HSPCs. In other embodiments, thepopulation of cells is about 0.1% to about 1%, about 1% to about 3%,about 3% to about 5%, about 10%-about 15%, about 15%-20%, about 20%-25%,about 25%-30%, about 30%-35%, about 35%-40%, about 40%-45%, about45%-50%, about 60%-70%, about 70%-80%, about 80%-90%, about 90%-95%, orabout 95% to about 100% HSPCs.

In particular embodiments, the population of cells is about 0.1% toabout 1%, about 1% to about 3%, about 3% to about 5%, about 10%-about15%, about 15%-20%, about 20%-25%, about 25%-30%, about 30%-35%, about35%-40%, about 40%-45%, about 45%-50%, about 60%-70%, about 70%-80%,about 80%-90%, about 90%-95%, or about 95% to about 100% HSPCs.

HSPCs in the therapeutic compositions of the invention can beautologous/autogeneic (“self”) or non-autologous (“non-self,” e.g.,allogeneic, syngeneic or xenogeneic) relative to a subject to which thetherapeutic composition is to be administered. “Autologous,” as usedherein, refers to cells from the same subject. “Allogeneic,” as usedherein, refers to cells of the same species that differ genetically tothe cell in comparison. “Syngeneic,” as used herein, refers to cells ofa different subject that are genetically identical to the cell incomparison. “Xenogeneic,” as used herein, refers to cells of a differentspecies to the cell in comparison. In particular embodiments, the HSPCsof the invention are allogeneic or autologous.

HSPCs for use in the methods of the present invention may be depleted ofmature hematopoietic cells such as T cells, B cells, NK cells, dendriticcells, monocytes, granulocytes, erythroid cells, and their committedprecursors from bone marrow aspirate, umbilical cord blood, or mobilizedperipheral blood (mobilized leukapheresis product). Mature, lineagecommitted cells are depleted by immunodepletion, for example, bylabeling solid substrates with antibodies that bind to a panel ofso-called “lineage” antigens: CD2, CD3, CD11b, CD14, CD15, CD16, CD19,CD56, CD123, and CD235a. A subsequent step can be performed to furtherpurify the population of cells, in which a substrate labeled withantibodies that bind to the CD34⁺ antigen are used to isolate primitivehematopoietic stem and progenitor cells. Kits are commercially availablefor purifying stem and progenitor cells from various cell sources and inparticular embodiments, these kits are suitable for use with the methodsof the present invention. Exemplary commercially available kits forpurifying stem and progenitor cells include, but are not limited toLineage (Lin) Depletion Kit (Miltenyi Biotec); CD34⁺ enrichment kit(Miltenyi Biotec); RosettaSep (Stem Cell Technologies).

In one embodiment, the amount of HSPCs in the therapeutic composition isat least 0.1×10⁵ cells, at least 0.5×10⁵ cells, at least 1×10⁵ cells, atleast 5×10⁵ cells, at least 10×10⁵ cells, at least 0.5×10⁶ cells, atleast 0.75×10⁶ cells, at least 1×10⁶ cells, at least 1.25×10⁶ cells, atleast 1.5×10⁶ cells, at least 1.75×10⁶ cells, at least 2×10⁶ cells, atleast 2.5×10⁶ cells, at least 3×10⁶ cells, at least 4×10⁶ cells, atleast 5×10⁶ cells, at least 10×10⁶ cells, at least 15×10⁶ cells, atleast 20×10⁶ cells, at least 25×10⁶ cells, or at least 30×10⁶ cells.

In a particular embodiment, the amount of HSPCs in the therapeuticcomposition is about 0.1×10⁵ cells to about 10×10⁵ cells; about 0.5×10⁶cells to about 5×10⁶ cells; about 1×10⁶ cells to about 3×10⁶ cells;about 1.5×10⁶ cells to about 2.5×10⁶ cells; or about 2×10⁶ cells toabout 2.5×10⁶ cells.

In a particular embodiment, the amount of HSPCs in the therapeuticcomposition is about 1×10⁶ cells to about 3×10⁶ cells; about 1.0×10⁶cells to about 5×10⁶ cells; about 1.0×10⁶ cells to about 10×10⁶ cells,about 10×10⁶ cells to about 20×10⁶ cells, about 10×10⁶ cells to about30×10⁶ cells, or about 20×10⁶ cells to about 30×10⁶ cells.

In another embodiment, the amount of HSPCs in the therapeuticcomposition is about 1×10⁶ cells to about 30×10⁶ cells; about 1.0×10⁶cells to about 20×10⁶ cells; about 1.0×10⁶ cells to about 10×10⁶ cells,about 2.0×10⁶ cells to about 30×10⁶ cells, about 2.0×10⁶ cells to about20×10⁶ cells, or about 2.0×10⁶ cells to about 10×10⁶ cells.

In a particular embodiment, the amount of HSPCs in the therapeuticcomposition is about 1×10⁶ HSPCs, about 2×10⁶ cells, about 5×10⁶ cells,about 7×10⁶ cells, about 10×10⁶ cells, about 15×10⁶ cells, about 17×10⁶cells, about 20×10⁶ cells about 25×10⁶ cells, or about 30×10⁶ cells.

In one embodiment, the amount of HSPCs in the therapeutic composition isthe amount of HSPCs in a partial or single cord of blood, or is at least0.1×10⁵ cells/kg of bodyweight, at least 0.5×10⁵ cells/kg of bodyweight,at least 1×10⁵ cells/kg of bodyweight, at least 5×10⁵ cells/kg ofbodyweight, at least 10×10⁵ cells/kg of bodyweight, at least 0.5×10⁶cells/kg of bodyweight, at least 0.75×10⁶ cells/kg of bodyweight, atleast 1×10⁶ cells/kg of bodyweight, at least 1.25×10⁶ cells/kg ofbodyweight, at least 1.5×10⁶ cells/kg of bodyweight, at least 1.75×10⁶cells/kg of bodyweight, at least 2×10⁶ cells/kg of bodyweight, at least2.5×10⁶ cells/kg of bodyweight, at least 3×10⁶ cells/kg of bodyweight,at least 4×10⁶ cells/kg of bodyweight, at least 5×10⁶ cells/kg ofbodyweight, at least 10×10⁶ cells/kg of bodyweight, at least 15×10⁶cells/kg of bodyweight, at least 20×10⁶ cells/kg of bodyweight, at least25×10⁶ cells/kg of bodyweight, or at least 30×10⁶ cells/kg ofbodyweight.

D. Methods of Preparing the Enhanced Cells of the Invention

The invention contemplates in part, methods of preparing HSPCscharacterized by increased levels of CXCR4 gene expression. In aparticular embodiment, the method of preparing the HSPCs comprisestreating HSPCs ex vivo with one or more agents capable of increasingCXCR4 gene expression in the contacted cells under conditions sufficientto increase CXCR4 gene expression at least 30, 40, 50, 60, 70, or 80fold in the contacted cells compared to non-contacted cells. In oneembodiment, method of preparing the HSPCs of the invention comprisestreating HSPCs ex vivo with a prostaglandin pathway agonist and aglucocorticoid.

As used herein, the terms “conditions sufficient,” or “under conditionssufficient,” refer to the conditions for treating the HSPCs with one ormore agents to increase CXCR4 gene expression in the cells to surprisingand unexpected levels compared to control, vehicle, or non-treatedcells.

Conditions include, but are not limited to the source of the cells, theagents used to treat the cells and concentrations of agent(s), the timethe cells are exposed to the agent(s), and the temperature of treatment.

1. Agents Useful in Preparing Enhanced Cells

As used herein, “agent” refers to a compound or molecule capable ofincreasing CXCR4 gene expression in HSPCs treated with the agent, andrefers to compounds that increase CXCR4 expression when used eitheralone or in combination with another compound or molecule. In particularembodiments of the invention, a combination of two or more agents thatact synergistically to increase CXCR4 gene expression in HSPCs treatedwith the combination is used in preparing the enhanced HSPCs. Particularagents include, for example, compounds capable of stimulating theprostaglandin pathway, e.g., prostaglandin pathway agonists, as well asglucocorticoids.

2. Prostaglandin Pathway Agonists

As used herein, the term “prostaglandin pathway agonist” refers to anagent that stimulates prostaglandin cell signaling pathways, includingan agent that stimulates the PGE₂R₂ and/or PGE₂R₄ cell signalingpathways, and increases CXCR4 gene expression in the cells. Illustrativeexamples of prostaglandin pathway agonists that are suitable for use inpreparing cells of the invention, include, but are not limited to, PGE₂,dmPGE₂, 15(S)-15-methyl PGE₂, 20-ethyl PGE₂,8-iso-16-cyclohexyl-tetranor PGE₂, and PGE₂ analogues. In certainembodiments, PGE₂R₂ and PGE₂R₄ agonists and analogues thereof are ofparticular interest, and in some embodiments, the agent preferentiallybinds and activates a PGE₂ EP₂ or PGE₂ EP₄ receptor.

As used herein, the terms “prostaglandin E₂” or “PGE₂” include, withoutlimitation, any naturally-occurring or chemically synthesized PGE₂molecule, as well as “analogues” thereof. As used herein, the term“analogue” or relates to a chemical molecule that is similar to anotherchemical substance, e.g., PGE₂, in structure and function, oftendiffering structurally by a single element or group, but may differ bymodification of more than one group (e.g., 2, 3, or 4 groups) if itretains the same function as the parental chemical. Such modificationsare routine to persons skilled in the art, and include, for example,additional or substituted chemical moieties, such as esters or amides ofan acid, protecting groups such as a benzyl group for an alcohol orthiol, and tert-butoxylcarbonyl groups for an amine. Also included aremodifications to alkyl side chains, such as alkyl substitutions (e.g.,methyl, dimethyl, ethyl, etc.), modifications to the level of saturationor unsaturation of side chains, and the addition of modified groups suchas substituted phenyl and phenoxy. Analogues can also includeconjugates, such as biotin or avidin moieties, enzymes such ashorseradish peroxidase and the like, and including radio-labeled,bioluminescent, chemoluminescent, or fluorescent moieties. Also,moieties may be added to the agents described herein to alter theirpharmacokinetic properties, such as to increase half-life in vivo or exvivo, or to increase their cell penetration properties, among otherdesirable properties. Also included are prodrugs, which are known toenhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) (see, e.g.,WO/2006/047476 for exemplary EP agonist prodrugs, which is incorporatedby reference for its disclosure of such agonists).

Illustrative examples of PGE₂ “analogues” include, without limitation,16,16-dimethyl PGE₂ (“dmPGE₂”), 16,16-dimethyl PGE₂p-(p-acetamidobenzamido) phenyl ester, 11-deoxy-16,16-dimethyl PGE₂,9-deoxy-9-methylene-16, 16-dimethyl PGE₂, 9-deoxy-9-methylene PGE₂,9-keto Fluprostenol, 5-trans PGE₂, 17-phenyl-omega-trinor PGE₂, PGE₂serinol amide, PGE₂ methyl ester, 16-phenyl tetranor PGE₂,15(S)-15-methyl PGE₂, 15(R)-15-methyl PGE₂, 8-iso-15-keto PGE₂, 8-isoPGE₂ isopropyl ester, 8-iso-16-cyclohexyl-tetranor PGE₂, 20-hydroxyPGE₂, 20-ethyl PGE₂, 11-deoxy PGEZ, nocloprost, sulprostone, butaprost,15-keto PGE₂, and 19 (R) hydroxy PGE₂. Also included are PG analogues orderivatives having a similar structure to PGE₂ that are substituted withhalogen at the 9-position (see, e.g., WO 2001/12596, herein incorporatedby reference in its entirety), as well as 2-decarboxy-2-phosphinicoprostaglandin derivatives, such as those described in U.S. PublicationNo. 2006/0247214, herein incorporated by reference in its entirety).

PGE₁ analogues, including without limitation alprostadil, can also beused to activate the PGE₂R₂ (EP₂) and PGE₂R₄ (EP₄) cell signalingpathways, and are contemplated as agents useful in the methods of theinvention.

Stimulation/activation of the PGE₂R₂ (EP₂) and PGE₂R₄ (EP₄) cellsignaling pathways are contemplated to underlie the physiologicalresponses in HSPCs that increase engraftment, maintain cell viability,and increase homing and proliferation of the cells. Accordingly, in oneembodiment, a “non-PGE₂-based ligand” that binds to and stimulatesPGE₂R₂ and PGE₂R₄ receptors (i.e., a PGE₂R₂/PGE₂R₄ agonist) iscontemplated for use in the methods of the invention.

Illustrative examples of non-PGE₂-based EP₂ receptor agonists includeCAY10399, ONO_8815Ly, ONO-AE1-259, CP-533,536 and carbazoles andfluorenes disclosed in WO 2007/071456.

Illustrative examples of non-PGE₂-based EP₄ agonists include ONO-4819,APS-999 Na, AH23848, ONO-AE1-329, and other non-PGE₂-based EP₄ agonistsdisclosed in WO/2000/038663; U.S. Pat. No. 6,747,037; and U.S. Pat. No.6,610,719).

Agents selective for the PGE₂ EP₄ receptor preferentially bind to andactivate PGE₂ EP₄ receptors. Such agents have a higher affinity for theEP₄ receptor than for any of the other three EP receptors namely EP₁,EP₂ and EP₃. Agents that selectively bind the PGE EP₄ receptor include,but are not limited to, agents selected from the group consisting of:5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone;2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxocyclopentyljsulfanylpropylsulfanyl]aceticacid; methyl4-[2-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]but-1-enyl]-5-oxocyclopentyl]ethylsulfanyl]butanoate;16-(3-Methoxymethyl)phenyl-ro-tetranor-5-thiaPGE;5-{3-[(2S)-2-{(3R)-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-5-oxopyrrolidin-1-yl]propyl]thiophene-2-carboxylate;[4′-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonicacid (3-methyl-thiophene-2-carbonyl)-amide]; and((Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoicacid), and pharmaceutically acceptable salts of any of these agents.

In particular embodiments, the prostaglandin pathway agonist is PGE₂,dmPGE₂, 15(S)-15-methyl PGE₂, 20-ethyl PGE₂, or8-iso-16-cyclohexyl-tetranor PGE₂.

3. Glucocorticoids

Illustrative examples of glucocorticoids and glucocorticoid receptoragonists suitable for use in the methods of the invention include, butare not limited to, medrysone, alclometasone, alclometasonedipropionate, amcinonide, beclometasone, beclomethasone dipropionate,betamethasone, betamethasone benzoate, betamethasone valerate,budesonide, ciclesonide, clobetasol, clobetasol butyrate, clobetasolpropionate, clobetasone, clocortolone, cloprednol, cortisol, cortisone,cortivazol, deflazacort, desonide, desoximetasone, desoxycortone,desoxymethasone, dexamethasone, diflorasone, diflorasone diacetate,diflucortolone, diflucortolone valerate, difluorocortolone,difluprednate, fluclorolone, fluclorolone acetonide, fludroxycortide,flumetasone, flumethasone, flumethasone pivalate, flunisolide,flunisolide hemihydrate, fluocinolone, fluocinolone acetonide,fluocinonide, fluocortin, fluocoritin butyl, fluocortolone,fluorocortisone, fluorometholone, fluperolone, fluprednidene,fluprednidene acetate, fluprednisolone, fluticasone, fluticasonepropionate, formocortal, halcinonide, halometasone, hydrocortisone,hydrocortisone acetate, hydrocortisone aceponate, hydrocortisonebuteprate, hydrocortisone butyrate, loteprednol, meprednisone,6a-methylprednisolone, methylprednisolone, methylprednisolone acetate,methylprednisolone aceponate, mometasone, mometasone furoate, mometasonefuroate monohydrate, paramethasone, prednicarbate, prednisolone,prednisone, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide and ulobetasol, as well as combinations thereof.

In particular embodiments, the glucocorticoid comprises medrysone,hydrocortisone, triamcinolone, alclometasone, or dexamethasone. In moreparticular embodiments, the glucocorticoid is medrysone.

4. Combinations of Agents

Combinations of agents can also be used in preparing the enhanced HSPCsof the invention, and in particular embodiments treating HSPCs with acombination of agents results in an unexpected synergistic increase inCXCR4 gene and protein expression in treated cells. In particular, HSPCstreated with combinations of prostaglandin pathway agonists andglucocorticoids exhibit an unexpectedly high increase in CXCR4 gene andprotein expression, and this correlates to improved therapeuticproperties of the treated cells compared to control, vehicle, ornon-treated cells.

In particular embodiments of the invention, the HSPCs are treated with acombination of one or more prostaglandin pathway agonists and one ormore glucocorticoids.

In particular embodiments of the invention, the prostaglandin pathwayagonist in the combination is a compound that selectively binds the PGE₂EP₂ or the PGE₂ EP₄ receptor. In other embodiments of the invention, theprostaglandin pathway agonist comprises PGE₂, or a PGE₂ analogue orderivative thereof. In particular embodiments, the prostaglandin pathwayagonist is PGE₂, dmPGE₂, 15(S)-15-methyl PGE₂, 20-ethyl PGE₂, or8-iso-16-cyclohexyl-tetranor PGE₂. In more particular embodiments of theinvention, the prostaglandin pathway agonist is PGE₂ or 16,16-dimethylPGE₂.

In some embodiments, the glucocorticoid in the combination is selectedfrom the group consisting of medrysone, alclometasone, alclometasonedipropionate, amcinonide, beclometasone, beclomethasone dipropionate,betamethasone, betamethasone benzoate, betamethasone valerate,budesonide, ciclesonide, clobetasol, clobetasol butyrate, clobetasolpropionate, clobetasone, clocortolone, cloprednol, cortisol, cortisone,cortivazol, deflazacort, desonide, desoximetasone, desoxycortone,desoxymethasone, dexamethasone, diflorasone, diflorasone diacetate,diflucortolone, diflucortolone valerate, difluorocortolone,difluprednate, fluclorolone, fluclorolone acetonide, fludroxycortide,flumetasone, flumethasone, flumethasone pivalate, flunisolide,flunisolide hemihydrate, fluocinolone, fluocinolone acetonide,fluocinonide, fluocortin, fluocoritin butyl, fluocortolone,fluorocortisone, fluorometholone, fluperolone, fluprednidene,fluprednidene acetate, fluprednisolone, fluticasone, fluticasonepropionate, formocortal, halcinonide, halometasone, hydrocortisone,hydrocortisone acetate, hydrocortisone aceponate, hydrocortisonebuteprate, hydrocortisone butyrate, loteprednol, meprednisone,6a-methylprednisolone, methylprednisolone, methylprednisolone acetate,methylprednisolone aceponate, mometasone, mometasone furoate, mometasonefuroate monohydrate, paramethasone, prednicarbate, prednisolone,prednisone, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide and ulobetasol.

In more particular embodiments, the glucocorticoid in the combinationcomprises medrysone, hydrocortisone, alclometasone, dexamethasone,methylprednisolone, or triamcinolone. In one embodiment, theglucocorticoid is medrysone.

In some embodiments, the HSPCs are treated with a combination comprisinga prostaglandin pathway agonist selected from the group consisting ofPGE₂, dmPGE₂, 15(S)-15-methyl PGE₂, 20-ethyl PGE₂, and8-iso-16-cyclohexyl-tetranor PGE₂ and one or more glucocorticoids. In aparticular embodiment, the HSPCs are treated with a combinationcomprising PGE₂ or dmPGE₂ and a glucocorticoid.

In some embodiments, the HSPCs are treated with a combination comprisinga prostaglandin pathway agonist selected from the group consisting ofPGE₂, dmPGE₂, 15(S)-15-methyl PGE₂, 20-ethyl PGE₂, and8-iso-16-cyclohexyl-tetranor PGE₂, and a glucocorticoid selected fromthe group consisting of medrysone, hydrocortisone, alclometasone,dexamethasone, methylprednisolone, or triamcinolone.

In other embodiments, the combination comprises PGE₂ or dmPGE₂ andmedrysone, hydrocortisone, alclometasone, dexamethasone,methylprednisolone, or triamcinolone. In more particular embodiments,the HSPCs are treated with a combination comprising PGE₂ or dmPGE₂ andmedrysone.

5. Formulations of Agents

Using cGMP practices, agents useful in preparing the therapeuticcomposition of the invention can be formulated in an organic solvent,such as methyl acetate, for use in contacting the cells of theinvention, and may be supplied in an endotoxin free vessel. Agentscontemplated by the invention are suitable for ex vivo administration tomammalian cells, as described herein. In certain embodiments, thesolvent is typically a suitable organic solvent, as described herein(e.g., DMSO, DMF, DME, etc., including combinations or mixturesthereof). One or more solvents may be combined at certain ratios. Forinstance, a mixture of two solvents may be combined at a ratio of9.5:0.5, 9:1, 8:2, 7:3, 6:4, 5:5, etc., including all integers anddecimal points.

The recitation “organic solvent” or “suitable organic solvent” relatesgenerally to carbon containing liquids or gases that dissolve a solid,liquid, or gaseous solute, resulting in a solution. A “suitable” organicsolvent is one that is appropriate for ex vivo administration to, orincubation with, mammalian cells, and may also be appropriate for invivo administration to a subject, such as by having minimal toxicity orother inhibitory effects under ex vivo conditions (e.g., cell culture)or in vivo at a selected concentration for the time of incubation oradministration. A suitable organic solvent should also be appropriatefor storage stability and handling of the agents described herein.Examples of suitable organic solvents include, but are not limited to,dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane(DME), and dimethylacetamide, including mixtures or combinationsthereof. In certain embodiments, a composition or organic solvent is“substantially free” of methyl acetate, meaning that there should be nomore than trace amounts of methyl acetate in the composition or solvent,and preferably undetectable amounts (e.g., as measured by high pressureliquid chromatography (HPLC), gas chromatography (GC), etc.).

As used herein, the term “endotoxin free” refers to vessels and/orcompositions that contain at most trace amounts (i.e., amounts having noadverse physiological effects to a subject) of endotoxin, and preferablyundetectable amounts of endotoxin. By “substantially free of endotoxin”is meant that there is less endotoxin per dose of cells than is allowedby the FDA for a biologic, which is a total endotoxin of 5 EU/kg bodyweight per day, which for an average 70 kg person is 350 EU per totaldose of cells. In one embodiment, the term “endotoxin free” refers to avessel and/or compositions that is at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% endotoxin free. Endotoxins aretoxins associated with certain bacteria, typically gram-negativebacteria, although endotoxins may be found in gram-positive bacteria,such as Listeria monocytogenes. The most prevalent endotoxins arelipopolysaccharides (LPS) or lipooligosaccharides (LOS) found in theouter membrane of various Gram-negative bacteria, and which represent acentral pathogenic feature in the ability of these bacteria to causedisease. Small amounts of endotoxin in humans can produce fever, alowering of the blood pressure, and activation of inflammation andcoagulation, among other adverse physiological effects. Therefore, it isoften desirable to remove most or all traces of endotoxin from drugproduct containers, because even small amounts may cause adverse effectsin humans. Endotoxins can be removed from vessels using methods known inthe art, for example, vessels can be cleaned in HEPA filtered washingequipment with endotoxin-free water, depyrogenated at 250° C., andclean-packaged in HEPA filtered workstations located inside a class100/10 clean room (e.g., a class 100 clean room, contains no more than100 particles bigger than half a micron in a cubic foot of air).

In particular embodiments, the HSPCs are treated (e.g., contacted) withone or more agents, each at a final concentration of about 1 μM to about100 μM. In certain embodiments, HSPCs are treated with one or moreagents, each at a final concentration of about 1×10⁻¹⁴ M to about 1×10⁻³M, about 1×10⁻¹³ M to about 1×10⁻⁴ M, about 1×10⁻¹² M to about 1×10⁻⁵ M,about 1×10⁻¹¹ M to about 1×10⁻⁴ M, about 1×10⁻¹¹ M to about 1×10⁻⁵ M,about 1×10⁻¹⁰ M to about 1×10⁻⁴ M, about 1×10⁻¹⁰ M to about 1×10⁻⁵ M,about 1×10⁻⁹ M to about 1×10⁻⁴ M, about 1×10⁻⁹ M to about 1×10⁻⁵ M,about 1×10⁻⁸ M to about 1×10⁻⁴ M, about 1×10⁻⁷ M to about 1×10⁻⁴ M,about 1×10⁻⁶ M to about 1×10⁻⁴ M, or any intervening ranges of finalconcentrations.

In another particular embodiment, HSPCs are treated with one or moreagents, each at a final concentration of about 1×10⁻¹⁴ M, about 1×10⁻¹³M, about 1×10⁻¹² M, about 1×10⁻¹⁰ M, about 1×10⁻⁹ M, about 1×10⁻⁸ M,about 1×10⁻⁷ M to about 1×10⁻⁶ M, about 1×10⁻⁵ M, about 1×10⁻⁴ M, about1×10⁻³ M, or any intervening final concentration. In treatmentscomprising one or more agents, the agents can be at differentconcentrations from each other or at the same concentration.

In particular embodiments, HSPCs are treated (e.g., contacted with oneor more agents) 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more times. The HSPCscan be intermittently, episodically, or sequentially contacted with oneor more agents within the same vessel (e.g., contacting the populationof cells with one drug for a period of time, exchanging the culturemedium and/or washing the population of cells, then repeating the cyclewith the same or a different combination of pharmaceutical agents forthe same predetermined period of time or a different predeterminedperiod of time).

6. Treatment of HSPCs

In one embodiment, the method of preparing the HSPCs comprises treatingHSPCs ex vivo with one or more agents capable of increasing CXCR4 geneexpression conditions sufficient to increase CXCR4 gene expression atleast 30, 40, 50, 60, 70, or 80 fold in the contacted cells compared tonon-contacted cells. The HSPCs may be treated with agents disclosedherein after isolation from a subject. In another embodiment, HSPCs areisolated from a subject and expanded prior to treatment with the agentsdisclosed herein. In one embodiment, the HSPCs are isolated from asubject and cryopreserved prior to treatment with the agents disclosedherein.

In particular embodiments, HSPCs are treated with one or more agents,e.g., a combination of a prostaglandin pathway agonist and aglucocorticoid, in an amount effective and for a time sufficient (i.e.,under conditions sufficient) to increase CXCR4 gene expression at least30, 40, 50, 60, 70, or 80 fold in the contacted cells compared tonon-contacted cells.

In various embodiments, sufficient temperature conditions includeincubation of the HSPCs with the one or more agents at a physiologicallyrelevant temperature, such as a temperature range of about 22° C. toabout 39° C. (about room temperature to about body temperature),including but not limited to temperatures of about 22° C., 23° C., 24°C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33°C., 34° C., 35° C., 36° C., 37° C., 38° C., and 39° C. In a particularembodiment, the sufficient temperature condition is between about 35° C.and 39° C. In one embodiment, the sufficient temperature condition isabout 37° C.

In various embodiments, a sufficient concentration of an agent is afinal concentration of about 10 nM to about 100 μM, about 100 nM, about500 nM, about 1 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM,about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 M, about100 μM, about 110 μM, or about 120 μM, or any other interveningconcentration of the agent (e.g., 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, 50μM, 100 μM). In a particular embodiment, the sufficient concentration ofeach agent is a final concentration of about 10 μM to about 25 μM. Inone embodiment, the sufficient concentration of an agent is a finalconcentration of about 10 μM.

In various embodiments, the sufficient time period for treating theHSPCs with one or more agents is an incubation period of about 60minutes to about 24 hours, about 60 minutes to about twelve hours, about60 minutes to about 6 hours, about 2 hours to about 6 hours, about 2hours to about 4 hours, and including, but not limited to, treatment fora duration of about 60 minutes, about 70 minutes, about 80 minutes,about 90 minutes, about 100 minutes, about 110 minutes, about 2 hours,about 2.5 hours, about 3 hours, about 3.5 hours or about 4 hours or anyother intervening duration. In a particular embodiment, the sufficientincubation period is about 2 hours to about 4 hours. In one embodiment,the sufficient incubation period for treating the HSPCs is about fourhours.

In particular embodiments, conditions sufficient to increase CXCR4 geneexpression at least 30, 40, 50, 60, 70, or 80 fold in the contactedcells compared to non-contacted cells comprises treating HSPCs ex vivoat a temperature range of about 22° C. to about 39° C.; at a finalconcentration of about 10 μM to about 25 μM of a prostaglandin pathwayagonist, and about 10 μM to about 25 μM of a glucocorticoid; andincubation with the agents for about 1 hour to about 4 hours, for about2 hours to about 3 hours, for about 2 hours to about 4 hours, or forabout 3 hours to about 4 hours.

In particular embodiments, conditions sufficient to increase CXCR4 geneexpression at least 30, 40, 50, 60, 70, or 80 fold in the contactedcells compared to non-contacted cells comprises treating HSPCs ex vivoat a temperature range of about 22° C. to about 39° C.; at a finalconcentration of about 10 μM to about 25 μM of PGE₂ or dmPGE₂, and about10 μM to about 25 μM of a glucocorticoid; and incubation with the agentsfor about 1 hour to about 4 hours, for about 2 hours to about 3 hours,for about 2 hours to about 4 hours, or for about 3 hours to about 4hours.

In particular embodiments, conditions sufficient to increase CXCR4 geneexpression at least 30, 40, 50, 60, 70, or 80 fold in the contactedcells compared to non-contacted cells comprises treating HSPCs ex vivoat a temperature range of about 22° C. to about 39° C.; at a finalconcentration of about 10 μM to about 25 μM of PGE₂ or dmPGE₂, and about10 μM to about 25 μM of a compound selected from the group consisting ofmedrysone, hydrocortisone, alclometasone, dexamethasone,methylprednisolone, or triamcinolone; and incubation with the agents(compounds) for about 1 hour to about 4 hours, for about 2 hours toabout 3 hours, for about 2 hours to about 4 hours, or for about 3 hoursto about 4 hours.

In particular embodiments, conditions sufficient to increase CXCR4 geneexpression at least 30, 40, 50, 60, 70, or 80 fold in the contactedcells compared to non-contacted cells comprises treating HSPCs ex vivoat a temperature range of about 22° C. to about 39° C.; at a finalconcentration of about 10 μM to about 25 μM of a prostaglandin pathwayagonist, and about 10 μM to about 25 μM of medrysone; and incubationwith the agents for about 1 hour to about 4 hours, for about 2 hours toabout 3 hours, for about 2 hours to about 4 hours, or for about 3 hoursto about 4 hours.

In another embodiment, conditions sufficient to increase CXCR4 geneexpression at least 30, 40, 50, 60, 70, or 80 fold in the contactedcells compared to non-contacted cells comprises treating HSPCs ex vivoinclude, incubation at a temperature of about 37° C. (about bodytemperature); a final concentration of about 10 μM PGE₂ or16,16-dimethyl PGE₂, in combination with a final concentration of about10 μM of a compound selected from the group consisting of medrysone,hydrocortisone, alclometasone, dexamethasone, methylprednisolone, ortriamcinolone; and incubation for about four hours.

In another embodiment, conditions sufficient to increase CXCR4 geneexpression at least 30, 40, 50, 60, 70, or 80 fold in the contactedcells compared to non-contacted cells comprises treating HSPCs ex vivoinclude, incubation at a temperature of about 37° C. (about bodytemperature); a final concentration of about 10 μM PGE₂ or16,16-dimethyl PGE₂, in combination with a final concentration of about10 μM of medrysone; and incubation for about four hours.

In particular embodiments, HSPCs are treated (e.g., contacted with oneor more agents) 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more times. Cells canbe intermittently, episodically, or sequentially contacted with one ormore agents within the same vessel (e.g., contacting the HSPCs with oneagent for a period of time, exchanging the culture medium and/or washingthe population of cells, then repeating the cycle with the same or adifferent combination of agents for the same predetermined period oftime or a different predetermined period of time).

E. Therapeutic Uses of the Enhanced Stem Cells

HSPCs described herein have increased CXCR4 gene and protein expression,and also exhibit enhanced therapeutic properties compared to untreatedcells. In particular, the HSPCs of the invention exhibit increasedhoming to bone marrow, ischemic tissue sites, and ischemia-damagedtissue, and also exhibit increased engraftment. The HSPCs of theinvention are thus useful for treating subjects in need of treatmentwith increased homing and/or engraftment of HSPCs to the bone marrow orsites of ischemia or ischemia-damaged tissue. In certain embodiments,the HSPCs are also useful for improving hematopoietic stem celltransplants and in treating ischemia or ischemia-damaged tissue, and inreducing further damage to ischemic tissue and/or repairing damage toischemic tissue through cell recruitment, improving vascularization inischemic tissue, improving tissue regeneration at sites of ischemia,decreasing ischemic tissue necrosis or apoptosis, and/or increasing cellsurvival at sites of ischemia. In particular embodiments, the HPSCs areuseful to subjects in need of hematopoietic reconstitution, such assubjects that have undergone or are scheduled to undergo myeloablativetherapy.

As used herein, the term “engraft” refers to the ability of a cell tointegrate into a location, such as a tissue, and persist in theparticular location over time. Cells may engraft in the bone marrow, forinstance, or in another location such as a site of injured or ischemictissue. “Homing” refers to the ability of HSPCs to localize, i.e.,travel, to a particular area or tissue. Homing may include localizationof administered HSPCs to the bone marrow or to another location such asa site of injured or ischemic tissue. In one embodiment, cells use achemoattractant mechanism to home to a particular tissue: cells havingincreased expression of CXCR4 have improved homing to ischemic tissuessecreting stromal cell derived factor 1 (SDF1), the cognate ligand ofCXCR4.

A “subject,” as used herein, includes any human that exhibits a symptomthat can be treated with a cell-based composition of the invention, orcan be treated with HSPCs having increased CXCR4 gene expression.

In various other embodiments, the invention provides methods of treatinga subject in need thereof that comprise identifying a subject in need,and administering to the subject HSPCs contacted with one or more agentsthat increase CXCR4 gene expression in the cells, including acombination of a prostaglandin pathway agonist and a glucocorticoid,under conditions sufficient to increase CXCR4 gene expression in thecells at least 30, 40, 50, 60, 70, or 80 fold compared to the level ofCXCR4 gene expression in non-contacted cells, thereby treating thesubject in need.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect,including without limitation achieving an improvement or elimination ofsymptoms of a disease. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of achieving an improvement or eliminationof symptoms, or providing a partial or complete cure for a diseaseand/or adverse affect attributable to the disease. “Treatment,” as usedherein, covers any treatment of a disease in a mammal, particularly in ahuman, and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; (c) relieving the disease, e.g., causing regression of thedisease, e.g., to completely or partially eliminate symptoms of thedisease; and (d) restoring the individual to a pre-disease state, e.g.,reconstituting the hematopoietic system.

“Treatment” or “treating,” as used herein, includes any desirable effecton the symptoms or pathology of a disease or pathological condition, andmay include even minimal reductions in one or more measurable markers ofthe disease or condition being treated. “Treatment” does not necessarilyindicate, or require, complete eradication or cure of the disease orcondition, or associated symptoms thereof.

1. Stem Cell Transplant Methods

“Subjects in need” of hematopoietic reconstitution, reconstitution ofthe hematopoietic system, and an increased number of HSPCs include, butare not limited to subjects that have or that have been diagnosed withvarious types of leukemias, anemias, lymphomas, myelomas, immunedeficiency disorders, and solid tumors as discussed elsewhere herein. A“subject” also includes a human who is a candidate for stem celltransplant or bone marrow transplantation, such as during the course oftreatment for a malignant disease or a component of gene therapy. Inparticular embodiments, a subject receives genetically modified HSPCs asa cell-based gene therapy. Subjects may also include individuals oranimals that donate stem cells or bone marrow for allogeneictransplantation. In certain embodiments, a subject may have undergonemyeloablative irradiation therapy or chemotherapy, or may haveexperienced an acute radiation or chemical insult resulting inmyeloablation. In certain embodiments, a subject may have undergoneirradiation therapy or chemotherapy, such as during various cancertreatments. Typical subjects include animals that exhibit aberrantamounts (lower or higher amounts than a “normal” or “healthy” subject)of one or more physiological activities that can be modulated by anagent or a stem cell or marrow transplant.

Subjects in need of hematopoietic reconstitution include subjectsundergoing chemotherapy or radiation therapy for cancer, as well assubjects suffering from (e.g., afflicted with) non malignant blooddisorders, particularly immunodeficiencies (e.g. SCID, Fanconi's anemia,severe aplastic anemia, or congenital hemoglobinopathies, or metabolicstorage diseases, such as Hurler's disease, Hunter's disease,mannosidosis, among others) or cancer, particularly hematologicalmalignancies, such as acute leukemia, chronic leukemia (myeloid orlymphoid), lymphoma (Hodgkin's or non-Hodgkin's), multiple myeloma,myelodysplastic syndrome, or non-hematological cancers such as solidtumors (including breast cancer, ovarian cancer, brain cancer, prostatecancer, lung cancer, colon cancer, skin cancer, liver cancer, orpancreatic cancer).

Subjects may also include subjects suffering from aplastic anemia, animmune disorder (severe combined immune deficiency syndrome or lupus),myelodysplasia, thalassemaia, sickle-cell disease or Wiskott-Aldrichsyndrome. In some embodiments, the subject suffers from a disorder thatis the result of an undesired side effect or complication of anotherprimary treatment, such as radiation therapy, chemotherapy, or treatmentwith a bone marrow suppressive drug, such as zidovadine, chloramphenicalor gangciclovir. Such disorders include neutropenias, anemias,thrombocytopenia, and immune dysfunction.

Other subjects may have disorders caused by an infection (e.g., viralinfection, bacterial infection or fungal infection) which causes damageto stem or progenitor cells of the bone marrow.

In addition, subject suffering from the following conditions can alsobenefit from treatment using HSPCs of the invention: lymphocytopenia,lymphorrhea, lymphostasis, erythrocytopenia, erthrodegenerativedisorders, erythroblastopenia, leukoerythroblastosis; erythroclasis,thalassemia, myelofibrosis, thrombocytopenia, disseminated intravascularcoagulation (DIC), immune (autoimmune) thrombocytopenic purpura (ITP),HIV inducted ITP, myelodysplasia; thrombocytotic disease,thrombocytosis, congenital neutropenias (such as Kostmann's syndrome andSchwachman-Diamond syndrome), neoplastic associated-neutropenias,childhood and adult cyclic neutropaenia; post-infective neutropaenia;myelodysplastic syndrome; neutropaenia associated with chemotherapy andradiotherapy; chronic granulomatous disease; mucopolysaccharidoses;Diamond Blackfan; Sickle cell disease; or Beta thalassemia major.

In a particular embodiment, the subject is a bone marrow donor who hasdonated bone marrow, is a bone marrow donor who has yet to donate bonemarrow, is a bone marrow donor transplant recipient, has hematopoieticprogenitor cells under environmental stress, has anemia, has a reducedlevel of immune cell function compared to a normal subject, or has animmune system deficiency.

In a certain embodiment, the subject has myeloma, non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic myeloid leukemia, chronicmyelogenous leukemia, chronic granulocytic leukemia, acute lymphoblasticleukemia, acute nonlymphoblastic leukemia, or pre-leukemia.

In particular embodiments, the subject is in need of gene therapy, suchas, for example, a hemoglobinopathy. As used herein, the term“hemoglobinopathy” or “hemoglobinopathic condition” includes anydisorder involving the presence of an abnormal hemoglobin molecule inthe blood. Examples of hemoglobinopathies included, but are not limitedto, hemoglobin C disease, hemoglobin sickle cell disease (SCD), sicklecell anemia, and thalassemias. Also included are hemoglobinopathies inwhich a combination of abnormal hemoglobins are present in the blood(e.g., sickle cell/Hb-C disease).

The term “sickle cell anemia” or “sickle cell disease” is defined hereinto include any symptomatic anemic condition which results from sicklingof red blood cells. Manifestations of sickle cell disease include:anemia; pain; and/or organ dysfunction, such as renal failure,retinopathy, acute-chest syndrome, ischemia, priapism and stroke. Asused herein the term “sickle cell disease” refers to a variety ofclinical problems attendant upon sickle cell anemia, especially in thosesubjects who are homozygotes for the sickle cell substitution in HbS.Among the constitutional manifestations referred to herein by use of theterm of sickle cell disease are delay of growth and development, anincreased tendency to develop serious infections, particularly due topneumococcus, marked impairment of splenic function, preventingeffective clearance of circulating bacteria, with recurrent infarcts andeventual destruction of splenic tissue. Also included in the term“sickle cell disease” are acute episodes of musculoskeletal pain, whichaffect primarily the lumbar spine, abdomen, and femoral shaft, and whichare similar in mechanism and in severity to the bends. In adults, suchattacks commonly manifest as mild or moderate bouts of short durationevery few weeks or months interspersed with agonizing attacks lasting 5to 7 days that strike on average about once a year. Among events knownto trigger such crises are acidosis, hypoxia and dehydration, all ofwhich potentiate intracellular polymerization of HbS (J. H. Jandl,Blood: Textbook of Hematology, 2nd Ed., Little, Brown and Company,Boston, 1996, pages 544-545). As used herein, the term “thalassemia”encompasses hereditary anemias that occur due to mutations affecting thesynthesis of hemoglobin. Thus, the term includes any symptomatic anemiaresulting from thalassemic conditions such as severe or β-thalassemia,thalassemia major, thalassemia intermedia, α-thalassemias such ashemoglobin H disease.

As used herein, “thalassemia” refers to a hereditary disordercharacterized by defective production of hemoglobin. Examples ofthalassemias include β and a thalassemia, β thalassemias are caused by amutation in the beta globin chain, and can occur in a major or minorform. In the major form of β thalassemia, children are normal at birth,but develop anemia during the first year of life. The mild form of βthalassemia produces small red blood cells a thalassemias are caused bydeletion of a gene or genes from the globin chain. As used herein,“antisickling proteins” include proteins which prevent or reverse thepathological events leading to sickling of erythrocytes in sickle cellconditions. In one embodiment of the invention, the transduced cells ofthe invention are used to deliver antisickling proteins to a subjectwith a hemoglobinopathic condition. Antisickling proteins also includemutated β-globin genes comprising antisickling amino acid residues.

In various embodiments, the invention provides, in part, methods forobtaining and preparing HSPCs for a hematopoietic stem progenitor celltransplant, comprising contacting HSPCs with one or more agents thatincrease CXCR4 gene expression in the HSPCs, including a combination ofa prostaglandin pathway agonist and a glucocorticoid, under conditionssufficient to increase CXCR4 gene expression in the HSPCs at least 30,40, 50, 60, 70, or 80 fold compared to the level of CXCR4 geneexpression in non-contacted cells.

In various other embodiments, the invention provides, in part, a methodof increasing hematopoietic stem and progenitor cell homing in a subjectcomprising contacting HSPCs with one or more agents that increase CXCR4gene expression in the HSPCs, including a combination of a prostaglandinpathway agonist and a glucocorticoid, under conditions sufficient toincrease CXCR4 gene expression in the HSPCs at least 30, 40, 50, 60, 70,or 80 fold compared to the level of CXCR4 gene expression innon-contacted cells,

In particular embodiments, the treated HSPCs are washed to substantiallyremove the agents, and subsequently administered to a subject in need ofan increase in hematopoietic stem cell homing.

The invention contemplates, in part, methods to increase stem cellengraftment in a subject in need thereof comprising contacting apopulation of cells that comprises HSPCs (e.g., bone marrow cells,peripheral blood cells, and/or umbilical cord blood cells) with one ormore agents that increase CXCR4 gene expression in the HSPCs, includinga combination of a prostaglandin pathway agonist and a glucocorticoid,under conditions sufficient to increase CXCR4 gene expression in theHSPCs at least 30, 40, 50, 60, 70, or 80 fold compared to the level ofCXCR4 gene expression in non-contacted cells, and administering theenhanced HSPCs to the subject.

In a particular embodiment, the invention provides a method of treatinga subject in need of hematopoietic reconstitution or reconstitution ofthe hematopoietic system comprising identifying a subject in need ofhematopoietic reconstitution, and administering to the subject an amountof HSPCs contacted with one or more agents that increase CXCR4 geneexpression in the HSPCs, including a combination of a prostaglandinpathway agonist and a glucocorticoid, under conditions sufficient toincrease CXCR4 gene expression in the HSPCs at least 30, 40, 50, 60, 70,or 80 fold compared to the level of CXCR4 gene expression innon-contacted cells, thereby treating the subject in need ofhematopoietic reconstitution.

In another particular embodiment, the invention provides a method oftreating a subject in need of hematopoietic reconstitution,reconstitution of the hematopoietic system, or an increased number ofHSPCs, comprising identifying a subject in need of hematopoieticreconstitution, and administering to the subject an amount of HSPCscontacted with one or more agents that increase CXCR4 gene expression inthe HSPCs, including a combination of a prostaglandin pathway agonistand a glucocorticoid, under conditions sufficient to increase CXCR4 geneexpression in the HSPCs at least 30, 40, 50, 60, 70, or 80 fold comparedto the level of CXCR4 gene expression in non-contacted cells, therebytreating the subject in need of hematopoietic reconstitution.

In another particular embodiment, the invention contemplates, a methodof treating a subject in need of a hematopoietic stem cell transplantthat comprises: selecting the subject in need of a hematopoietic stemcell transplant and administering to a subject HSPCs that have beencontacted with one or more agents that increase CXCR4 gene expression inthe cells, including a combination of a prostaglandin pathway agonistand a glucocorticoid, under conditions sufficient to increase CXCR4 geneexpression in the HSPCs at least 30, 40, 50, 60, 70, or 80 fold comparedto the level of CXCR4 gene expression in non-contacted cells. Inparticular embodiments, the subject is in need of hematopoieticreconstitution.

In particular illustrative embodiments of the methods described hereinfor increasing homing or engraftment of HSPCs, or for treating subjectsin need of hematopoietic reconstitution, reconstitution of thehematopoietic system, or for performing a hematopoietic stem celltransplant, the HSPCs are treated with a combination of one or moreagents that includes (i) PGE₂, dmPGE₂, 15(S)-15-methyl PGE₂, 20-ethylPGE₂, or 8-iso-16-cyclohexyl-tetranor PGE₂ and (ii) a glucocorticoid. Inmore particular embodiments, the combination includes (i) PGE₂ or16,16-dimethyl PGE₂ and (ii) medrysone, hydrocortisone, dexamethasone,methylprednisolone, triamcinolone, or alclometasone. In more particularembodiments, the combination includes (i) PGE₂ or 16,16-dimethyl PGE₂and (ii) medrysone.

Without wishing to be bound to any particular theory, the presentinvention contemplates, in part, that one of the advantages of using theenhanced HSPCs of the invention in stem cell transplants is that fewerHSPCs can be used in a transplant because the enhanced HSPCs have, forexample, increased engraftment potential, improved homing, and increasedcapacity for in vivo expansion compared to untreated HSPCs.

2. Ischemic Tissue Treatment Methods

The invention provides methods of cell-based therapy for treatingischemic tissue or treating or ameliorating one or more symptomsassociated with tissue ischemia, including, but not limited to,impaired, or loss of, organ function (including without limitationimpairments or loss of brain, kidney, or heart function), cramping,claudication, numbness, tingling, weakness, pain, reduced wound healing,inflammation, skin discoloration, and gangrene.

Ischemic tissue may be treated by increased homing of stem cells tosites of ischemic tissue damage, increased recruitment of endogenousstem cells and endothelial progenitor cells at the ischemic tissue site,increased vascularization at the ischemic tissue site, reducing ischemictissue necrosis or programmed cell death, or increasing cell survival atthe ischemic tissue site. Accordingly, the invention contemplates, inpart, cells having these therapeutic properties would be useful intreating ischemic tissue or a tissue damaged by ischemia or treating orameliorating at least one symptom associated with an ischemic tissue.

As used herein, the terms “ischemia,” “ischemic condition,” or “ischemicevent” mean any decrease or stoppage in the blood supply to any cell,tissue, organ, or body part caused by any constriction, damage, orobstruction of the vasculature. Ischemia sometimes results fromvasoconstriction or thrombosis or embolism. Ischemia can lead to directischemic injury, tissue damage due to cell death caused by reducedsupply of oxygen (hypoxia, anoxia), glucose, and nutrients. “Hypoxia” ora “hypoxic condition” intends a condition under which a cell, organ ortissue receives an inadequate supply of oxygen. “Anoxia” refers to avirtually complete absence of oxygen in the organ or tissue, which, ifprolonged, may result in death of the cell, organ or tissue.

“Symptoms associated with ischemia,” “symptoms resulting from ischemia,”or “symptoms caused by ischemia” refers to symptoms that include, butare not limited to: impaired, or loss of, organ function (includingwithout limitation impairments or loss of brain, kidney, or heartfunction), cramping, claudication, numbness, tingling, weakness, pain,reduced wound healing, inflammation, skin discoloration, and gangrene.

“Ischemic tissue injury,” “ischemic tissue damage,” “tissue damage dueto ischemia,” “tissue damage associated with ischemia,” “tissue damageas a result of ischemia,” “tissue damaged caused by ischemia,” and“ischemic-damaged tissue” refers to morphological, physiological, and/ormolecular damage to an organ or tissue or cell as a result of a periodof ischemia.

In one embodiment, the subject exhibits at least one symptom of anischemic tissue or tissue damaged by ischemia. In particularembodiments, the subject is a human who is has or who is at risk ofhaving an ischemic tissue or tissue damaged by ischemia, e.g., a subjectthat has diabetes, peripheral vascular disease, thromboangiitisobliterans, vasculitis, cardiovascular disease, coronary artery diseaseor heart failure, or cerebrovascular disease, cardiovascular disease, orcerebrovascular disease.

The invention also provides, in particular embodiments, a method oftreating ischemic tissue or a tissue damaged by ischemia, comprisingadministering to a patient in need of such treatment HSPCs contactedwith one or more agents that increase CXCR4 gene expression in thecells, including a combination of a prostaglandin pathway agonist and aglucocorticoid, under conditions sufficient to increase CXCR4 geneexpression in the cells at least 30, 40, 50, 60, 70, or 80 fold comparedto the level of CXCR4 gene expression in non-contacted cells.

In one embodiment, the cells provide therapy to the subject by increasedhoming of stem cells to sites of ischemic tissue damage, increasedrecruitment of endogenous stem cells and endothelial progenitor cells atthe ischemic tissue site, increased stimulation of vascularization atthe ischemic tissue site, reducing ischemic tissue necrosis orprogrammed cell death, or increasing cell survival at the ischemictissue site.

In various other embodiments, the invention provides a method oftreating or ameliorating an ischemic tissue injury comprisingadministering to a subject a therapeutically effective amount of acomposition comprising HSPCs contacted with one or more agents thatincrease CXCR4 gene expression in the cells, including a combination ofa prostaglandin pathway agonist and a glucocorticoid, under conditionssufficient to increase CXCR4 gene expression in the cells at least 30,40, 50, 60, 70, or 80 fold compared to the level of CXCR4 geneexpression in non-contacted cells.

In various other embodiments, the invention provides a method oftreating or ameliorating a symptom associated with an ischemic tissueinjury comprising administering to a subject HSPCs contacted with one ormore agents that increase CXCR4 gene expression in the cells, includinga combination of a prostaglandin pathway agonist and a glucocorticoid,under conditions sufficient to increase CXCR4 gene expression in thecells at least 30, 40, 50, 60, 70, or 80 fold compared to the level ofCXCR4 gene expression in non-contacted cells.

Illustrative examples of tissues that are suitable for treatment withthe compositions of the present invention include, mesodermal tissue,endodermal tissue, or ectodermal tissue. Other tissues suitable fortreatment with the compositions of the present invention include, butare not limited to, skin tissue, skeletal muscle tissue, cardiac muscletissue, smooth muscle tissue, cartilage tissue, tendon tissue, bonetissue, brain tissue, spinal cord tissue, retinal tissue, cornealtissue, lung tissue, liver tissue, kidney tissue, pancreatic tissue,ovarian tissue, testicular tissue, intestinal tissue, stomach tissue,and bladder tissue.

In particular embodiments, any tissue that has a compromised bloodsupply and is ischemic or at risk for becoming ischemic may be treatedusing the methods of the invention.

Illustrative examples of genetic disorders, syndromic conditions,traumatic injuries, chronic conditions, medical interventions, or otherconditions that cause or are associated with ischemia, or increase therisk of ischemia in a subject, or cause a subject to exhibit more ormore symptoms of ischemia, and thus, suitable for treatment oramelioration using the methods of the present invention, include, butare not limited to, acute coronary syndrome, acute lung injury (ALI),acute myocardial infarction (AMI), acute respiratory distress syndrome(ARDS), arterial occlusive disease, arteriosclerosis, articularcartilage defect, aseptic systemic inflammation, atheroscleroticcardiovascular disease, autoimmune disease, bone fracture, bonefracture, brain edema, brain hypoperfusion, Buerger's disease, burns,cancer, cardiovascular disease, cartilage damage, cerebral infarct,cerebral ischemia, cerebral stroke, cerebrovascular disease,chemotherapy-induced neuropathy, chronic infection, chronic mesentericischemia, claudication, congestive heart failure, connective tissuedamage, contusion, coronary artery disease (CAD), critical limb ischemia(CLI), Crohn's disease, deep vein thrombosis, deep wound, delayed ulcerhealing, delayed wound-healing, diabetes (type I and type II), diabeticneuropathy, diabetes induced ischemia, disseminated intravascularcoagulation (DIC), embolic brain ischemia, graft-versus-host disease,frostbite, hereditary hemorrhagic telengiectasiaischemic vasculardisease, hyperoxic injury, hypoxia, inflammation, inflammatory boweldisease, inflammatory disease, injured tendons, intermittentclaudication, intestinal ischemia, ischemia, ischemic brain disease,ischemic heart disease, ischemic peripheral vascular disease, ischemicplacenta, ischemic renal disease, ischemic vascular disease,ischemic-reperfusion injury, laceration, left main coronary arterydisease, limb ischemia, lower extremity ischemia, myocardial infarction,myocardial ischemia, organ ischemia, osteoarthritis, osteoporosis,osteosarcoma, Parkinson's disease, peripheral arterial disease (PAD),peripheral artery disease, peripheral ischemia, peripheral neuropathy,peripheral vascular disease, pre-cancer, pulmonary edema, pulmonaryembolism, remodeling disorder, renal ischemia, retinal ischemia,retinopathy, sepsis, skin ulcers, solid organ transplantation, spinalcord injury, stroke, subchondral-bone cyst, thrombosis, thrombotic brainischemia, tissue ischemia, transient ischemic attack (TIA), traumaticbrain injury, ulcerative colitis, vascular disease of the kidney,vascular inflammatory conditions, von Hippel-Lindau syndrome, and woundsto tissues or organs.

Other illustrative examples of genetic disorders, syndromic conditions,traumatic injuries, chronic conditions, medical interventions, or otherconditions that cause or are associated with ischemia, or increase therisk of ischemia in a subject, or cause a subject to exhibit more ormore symptoms of ischemia suitable for treatment or amelioration usingthe methods of the present invention, include, ischemia resulting fromsurgery, chemotherapy, radiation therapy, or cell, tissue, or organtransplant or graft.

In various embodiments, the methods of the invention are suitable fortreating cerebrovascular ischemia, myocardial ischemia, limb ischemia(CLI), myocardial ischemia (especially chronic myocardial ischemia),ischemic cardiomyopathy, cerebrovascular ischemia, renal ischemia,pulmonary ischemia, intestinal ischemia, and the like.

In various embodiments, the invention contemplates that the therapeuticcell compositions disclosed herein can be used to treat an ischemictissue in which it is desirable to increase the blood flow, oxygensupply, glucose supply, or supply of nutrients to the tissue.

3. Expanded HSPCs

The invention further contemplates that the enhanced HSPCs provided bythe invention are not expanded ex vivo or in vitro prior toadministration to a subject. In particular embodiments, an unexpandedpopulation of HSPCs is obtained, the population of HSPCs is treated exvivo in accordance with the protocol provided herein to obtain enhancedHSPCS, the enhanced HSPCs may be washed to remove the treatmentagent(s), and the enhanced HSPCs are administered to a patient withoutexpansion of the HSPC population ex vivo. In some embodiments, HSPCs areobtained from a donor, including cord blood, and are not expanded priorto or after treatment of the HSPCs, or at any time prior toadministration of the therapeutic composition to a patient.

In one embodiment, an unexpanded population of HSPCs is treated and isadministered to a patient prior to any substantial ex vivo cell divisionof the HSPCs in the population, or prior to the time required for anysubstantial cell division ex vivo. In other embodiments, an unexpandedpopulation of HSPCs is treated and is administered to a patient prior toany substantial ex vivo mitosis of the HSPCs in the population, or priorto the time required for any substantial mitosis ex vivo. In someembodiments, an unexpanded population of HSPCs is treated and isadministered to a patient prior to the doubling time of the HSPCs in thepopulation. In some embodiments, an unexpanded population of HSPCs istreated and is administered to a patient within 6, 12, or 24 hours oftreatment of the HSPCs. In other embodiments, an unexpanded populationof HSPCs is treated and is administered to a patient within 2 hours oftreatment of the HSPCs.

In various embodiments, the HSPCs of the invention are not culturedprior to treatment with one or more agents, or combinations of agents,ex vivo or at any time prior to administration to a patient. In someembodiments, the HSPCs are cultured for less than about 24 hours. Inother embodiments, the HSPCs are cultured for less than about 12 hours,10 hours, 8 hours, 6 hours, 4 hours, or two hours.

In other embodiments, the invention provides HSPCs that are expandedprior to treatment of the HSPCs with agents to obtain enhanced HSPCs.HSPCs, whether obtained from cord blood, bone marrow, peripheral blood,Wharton's jelly, placental blood or other source, may be grown orexpanded in any suitable, commercially available or custom definedmedium, with or without serum, as desired (see, e.g., Hartshorn et al.,Cell Technology for Cell Products, pages 221-224, R. Smith, Editor;Springer Netherlands, 2007, herein incorporated by reference in itsentirety). For instance, in certain embodiments, serum free medium mayutilize albumin and/or transferrin, which have been shown to be usefulfor the growth and expansion of CD34⁺ cells in serum free medium. Also,cytokines may be included, such as Flt-3 ligand, stem cell factor (SCF),and thrombopoietin (TPO), among others. HSPCs may also be grown invessels such as bioreactors (see, e.g., Liu et al., Journal ofBiotechnology 124:592-601, 2006, herein incorporated by reference in itsentirety). A suitable medium for ex vivo expansion of HSPCs may alsocomprise supporting cells, such as stromal cells (e.g., lymphoreticularstromal cells), which can be derived, for instance, from thedisaggregation of lymphoid tissue, and which have been show to supportthe in vitro, ex vivo, and in vivo maintenance, growth, anddifferentiation of hematopoietic stem and progenitor cells, as well astheir progeny.

In various embodiments, the enhanced HSPCs administered to a subject area heterogeneous population of cells including, whole bone marrow,umbilical cord blood, mobilized peripheral blood, hematopoietic stemcells, hematopoietic progenitor cells, and the progeny of hematopoieticstem and progenitor cells, including granulocytes (e.g., promyelocytes,myelocytes, metamyelocytes, neutrophils, eosinophils, basophils),erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g.,megakaryoblasts, platelet producing megakaryocytes, platelets), andmonocytes (e.g., monocytes, macrophages).

4. Administration of HSPCs and Compositions Thereof

In various embodiments, the present invention provides, in part, methodscomprising administration of treated HPSCs to a subject in need thereof.Suitable methods for administering populations of cells used in themethods described herein include parenteral administration, including,but not limited to methods of intravascular administration, such asintravenous and intraarterial administration. Additional illustrativemethods for administering cells of the invention include intramuscular,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

In one embodiment, a method for increasing homing or engraftment ofHSPCs comprises parenteral administration of HSPCs contacted with one ormore agents that increase CXCR4 gene expression in the cells, includinga combination of a prostaglandin pathway agonist and a glucocorticoid,under conditions sufficient to increase CXCR4 gene expression in thecells at least 30, 40, 50, 60, 70, or 80 fold compared to the level ofCXCR4 gene expression in non-contacted cells.

In one embodiment, a method for hematopoietic reconstitution,reconstitution of the hematopoietic system, or for performing ahematopoietic stem cell transplant, comprises parenteral administrationof HSPCs contacted with one or more agents that increase CXCR4 geneexpression in the cells, including a combination of a prostaglandinpathway agonist and a glucocorticoid, under conditions sufficient toincrease CXCR4 gene expression in the cells at least 30, 40, 50, 60, 70,or 80 fold compared to the level of CXCR4 gene expression innon-contacted cells.

In preferred embodiments, the HSPCs are administered or infused to asubject intravenously.

In particular illustrative embodiments of the methods described hereinfor increasing homing or engraftment of HSPCs, or for treating subjectsin need of hematopoietic reconstitution, reconstitution of thehematopoietic system, or for performing a hematopoietic stem celltransplant, comprise intravenously administering or infusing HSPCstreated with a combination of one or more agents that includes (i) PGE₂,dmPGE₂, 15(S)-15-methyl PGE₂, 20-ethyl PGE₂, or8-iso-16-cyclohexyl-tetranor PGE₂ and (ii) a glucocorticoid. In moreparticular embodiments, the methods comprise intravenously administeringor infusing HSPCs treated with (i) PGE₂ or 16,16-dimethyl PGE₂ and (ii)medrysone, hydrocortisone, dexamethasone, methylprednisolone,triamcinolone, or alclometasone. In more particular embodiments, themethods comprise intravenously administering or infusing HSPCs treatedwith (i) PGE₂ or 16,16-dimethyl PGE₂ and (ii) medrysone.

In particular embodiment, the composition may be administered to anindividual having ischemia, ischemic tissue, or at least one symptom ofischemia. Most preferably, the site of administration is close to ornearest the intended site of activity, i.e., near the site of tissueischemia. In cases when a subject suffers from global ischemia, asystemic administration, such as intravenous administration, ispreferred. Without intending to be bound by mechanism, when thetherapeutic compositions are administered, the HSPCs migrate or home tothe ischemic tissue in response to chemotactic factors produced due tothe injury to effect treatment of ischemic tissue or treatment andamelioration of at least one symptom associated with the ischemictissue.

The HSPCs can be injected directly into the area of ischemia, or thestem cells may be infused into an artery supplying the area of tissueischemia. Where the subject has a totally occluded vessel that wouldnormally supply the area of the ischemic tissue, the selected artery forinfusion is preferably a vessel that provides collateral flow to theischemic tissue in the distribution of the totally occluded vessel.

HSPCs and therapeutic compositions of the invention may be inserted intoa delivery device which facilitates introduction by injection orimplantation into the subjects. Such delivery devices may include tubes,e.g., catheters, for injecting cells and fluids into the body of arecipient subject. In one embodiment, the tubes additionally have aneedle, e.g., a syringe, through which the cells of the invention can beintroduced into the subject at a desired location. In a particularembodiment, cells are formulated for administration into a blood vesselvia a catheter (where the term “catheter” is intended to include any ofthe various tube-like systems for delivery of substances to a bloodvessel).

In one embodiment, a method for treating an ischemic tissue, or a tissuedamaged by ischemia, comprises parenteral administration of HSPCscontacted with one or more agents that increase CXCR4 gene expression inthe cells, including a combination of a prostaglandin pathway agonistand a glucocorticoid, under conditions sufficient to increase CXCR4 geneexpression in the cells at least 30, 40, 50, 60, 70, or 80 fold comparedto the level of CXCR4 gene expression in non-contacted cells.

In one embodiment, a method for treating or ameliorating at least onesymptom associated with an ischemic tissue or a tissue damaged byischemia, comprises parenteral administration of HSPCs contacted withone or more agents that increase CXCR4 gene expression in the cells,including a combination of a prostaglandin pathway agonist and aglucocorticoid, under conditions sufficient to increase CXCR4 geneexpression in the cells at least 30, 40, 50, 60, 70, or 80 fold comparedto the level of CXCR4 gene expression in non-contacted cells.

In preferred embodiments, the HSPCs are administered intravenously or bydirect injection to the ischemic site.

In particular illustrative embodiments of the methods described hereinfor treating or ameliorating ischemia or at least one symptom ofischemia, comprise intravenously administering or directly injectingHSPCs treated with a combination of one or more agents that includes (i)PGE₂, dmPGE₂, 15(S)-15-methyl PGE₂, 20-ethyl PGE₂, or8-iso-16-cyclohexyl-tetranor PGE₂ and (ii) a glucocorticoid. In moreparticular embodiments, the methods comprise intravenously administeringor infusing HSPCs treated with (i) PGE₂ or 16,16-dimethyl PGE₂ and (ii)medrysone, hydrocortisone, dexamethasone, methylprednisolone,triamcinolone, or alclometasone. In more particular embodiments, themethods comprise intravenously administering or infusing HSPCs treatedwith (i) PGE₂ or 16,16-dimethyl PGE₂ and (ii) medrysone.

In particular embodiments, the composition may be administered topicallyto a site of ischemic tissue damage, such as, for example, the surfaceof a wound, e.g., a non-healing wound, an ulcer, a burn, or frostbite.

The compositions of the invention may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: (1) parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation as partof a cell grafts, biocompatible scaffolds, etc. In various embodiments,a biocompatible scaffold or graft is provided to promote repair,replacement, and/or regeneration of a damaged, injured, or diseasedtissue or organ, e.g., an ischemic tissue.

In certain illustrative embodiments, a method for treating a subject inneed of the HPSCs of the invention comprises providing an biocompatiblescaffold or cell graft comprising HPSCs of the invention. As usedherein, the term “biocompatible scaffold” or “cell graft” refers to abiocompatible natural and/or synthetic structure comprising one or morecell-based compositions, cells, tissues, polymers, polynucleotides,lattices, and/or matrices that is injected, applied to the surface of,or engrafted within a patient or subject that is suitable for directingor attracting a cell-based composition to repair, regenerate, or replacea cell, tissue or organ in vivo.

In particular illustrative embodiments, an implant comprises abiocompatible matrix that can be molded into any suitable form and hasespecially important roles to prepare tissues in a three-dimensionalshape having a certain depth or height or a flat sheet-like shape forapplication to dermal wounds. Biomaterial science is an established andevolving field (Takayama et al., Principles of Tissue Engineering,Second Edition, edit Lanza R P, Langer R, Vacanti J., Academic Press,San Diego, 2000, pg 209-218; Saltmann et al., Principles of TissueEngineering, Second Edition, edit Lanza R P, Langer R, Vacanti J.,Academic Press, San Diego, 2000, p 221-236; Hubbell et al, Principles ofTissue Engineering, Second Edition, edit Lanza R P, Langer R, VacantiJ., Academic Press, San Diego, 2000, p 237-250; Thomson et al,Principles of Tissue Engineering, Second Edition, edit Lanza R P, LangerR, Vacanti J., Academic Press, San Diego, 2000, p 251-262; Pachence etal, Principles of Tissue Engineering, Second Edition, edit Lanza R P,Langer R, Vacanti J., Academic Press, San Diego, 2000, p 263-278).

Chemists have developed methods to synthesize biocompatible scaffoldcomprising polymers to direct and modulate cell growth in vitro, exvivo, and in vivo. The physical properties of the polymers can bemodulated to create solid and liquid matrices of specific strengths andviscosities. Some polymers are stable in vivo and will remain in apatient's body for up to 1, 2, 3, 4, 5, 10, 15 or more years. Otherpolymers are also biodegradable, resorbing at a fixed rate over time toallow replacement by newly synthesized extracellular matrix proteins.Resorption can occur within days to weeks or months followingimplantation (Pachence et al., Principles of Tissue Engineering, SecondEdition, edit Lanza R P, Langer R, Vacanti J., Academic Press, SanDiego, 2000, p 263-278).

In other illustrative embodiments, a biocompatible scaffold comprises abioabsorbable material. A porous carrier is preferably made of onecomponent or a combination of multiple components selected from thegroup consisting of collagen, collagen derivatives, hyaluronic acid,hyaluronates, chitosan, chitosan derivatives, polyrotaxane, polyrotaxanederivatives, chitin, chitin derivatives, gelatin, fibronectin, heparin,laminin, and calcium alginate; wherein a support member is made of onecomponent or a combination of multiple components selected from thegroup consisting of polylactic acid, polyglycolic acid,polycaprolactone, polylactic acid-polyglycolic acid copolymer,polylactic acid-polycaprolactone copolymer, and polyglycolicacid-polycaprolactone copolymer (see, for example, U.S. Pat. No.5,077,049 and U.S. Pat. No. 5,42,033, and U.S. Patent ApplicationPublication No. 2006/0121085, of which the polymer formulations andmethods of making the same of each patent and application isincorporated herein in its entirety).

In particular illustrative embodiments of the invention, thebiocompatible scaffold or cell graft comprises a viscous, biocompatibleliquid material. The biocompatible liquid is capable of gelling at bodytemperature and is selected from the group consisting of alginate,collagen, fibrin, hyaline, or plasma. The viscous, biocompatible liquidmaterial can also be combined with a malleable, three dimensional matrixcapable of filling an irregular tissue defect. The matrix is a materialincluding, but not limited to, polyglycolic-polylactic acid,poly-glycolic acid, poly-lactic acid, or suture-like material.

In further illustrative embodiments, biocompatible scaffolds or cellgrafts comprising matrices can be molded into desired shapes (e.g.,two-dimensional or three-dimensional structures) conducive to orfacilitating cell, tissue, and/or organ development. The implant can beformed from polymeric material, having fibers such as a mesh or sponge.Such a structure provides sufficient area on which the cells can growand proliferate. Desirably, the matrices of the scaffolds or cell graftsare biodegradable over time, so that they will be absorbed into theanimal matter as it develops. Suitable polymers can be homopolymers orheteropolymers and can be formed from monomers including, but notlimited to glycolic acid, lactic acid, propyl fumarate, caprolactone,and the like. Other suitable polymeric material can include a protein,polysaccharide, polyhydroxy acid, polyorthoester, polyanhydride,polyphosphozene, or a synthetic polymer, particularly a biodegradablepolymer, or any combination thereof.

Sheet-like scaffolds and grafts provide reparative, replacement, and/orregenerative therapy for dermal tissues, membranes for tooth rootcoverage procedures, membranous tissues (e.g., dura mater), flat bones(e.g., skull, breast-bone) and the like. Tubular implants and graftsprovide reparative, replacement, and/or regenerative therapy forarteries, veins, ureters, urethras, nerves, long bones (e.g., femur,fibula, tibia, humerus, radius, ulna, metacarpals, metatarsals, etc.)and the like. Other three dimensional implants and grafts providereparative, replacement, and/or regenerative therapy for organtransplants (e.g., liver, lung, skin, heart, pancreas, etc.), boneremodeling or mending of all types of bones, dental implants, or formuscle, tendon, ligament, and cartilage grafts.

In one embodiment, a method for treating or ameliorating at least onesymptom associated with an ischemic tissue or a tissue damaged byischemia, comprises direct administration, to an ischemic tissue, of abiocompatible scaffold or cell graft comprising HSPCs contacted with oneor more agents that increase CXCR4 gene expression in the cells,including a combination of a prostaglandin pathway agonist and aglucocorticoid, under conditions sufficient to increase CXCR4 geneexpression in the cells at least 30, 40, 50, 60, 70, or 80 fold comparedto the level of CXCR4 gene expression in non-contacted cells.

In particular illustrative embodiments of the methods described hereinfor treating or ameliorating at least one symptom associated with anischemic tissue or a tissue damaged by ischemia, comprise directadministration, to an ischemic tissue, of a biocompatible scaffold orcell graft comprising HSPCs treated with a combination of one or moreagents that includes (i) PGE₂, dmPGE₂, 15(S)-15-methyl PGE₂, 20-ethylPGE₂, or 8-iso-16-cyclohexyl-tetranor PGE₂ and (ii) a glucocorticoid. Inmore particular embodiments, the methods comprises directadministration, to an ischemic tissue, of a biocompatible scaffold orcell graft comprising HPSCs treated with (i) PGE₂ or 16,16-dimethyl PGE₂and (ii) medrysone, hydrocortisone, dexamethasone, methylprednisolone,triamcinolone, or alclometasone. In more particular embodiments, themethod comprises direct administration, to an ischemic tissue, of abiocompatible scaffold or cell graft comprising HPSCs treated with (i)PGE₂ or 16,16-dimethyl PGE₂ and (ii) medrysone.

F. Administration-Ready Compositions of the Invention

The compositions of treated cells of the invention are sterile, and aresuitable and ready for administration (i.e., can be administered withoutany further processing) to human patients. In some embodiments, thetherapeutic composition is ready for infusion into a patient. As usedherein, the terms “administration-ready,” “ready for administration” or“ready for infusion” refer to a cell based composition of the inventionthat does not require any further treatment or manipulations prior totransplant or administration to a subject.

The sterile, therapeutically acceptable compositions suitable foradministration to a patient may comprise one or more pharmaceuticallyacceptable carriers (additives) and/or diluents (e.g., pharmaceuticallyacceptable medium, for example, cell culture medium), or otherpharmaceutically acceptable components. Pharmaceutically acceptablecarriers and/or diluents are determined in part by the particularcomposition being administered, as well as by the particular method usedto administer the therapeutic composition. Accordingly, there is a widevariety of suitable formulations of therapeutic compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed. 1985)).

In particular embodiments, therapeutic cell compositions comprising stemand/or progenitor cells comprise a pharmaceutically acceptable cellculture medium. A therapeutic composition comprising a cell-basedcomposition of the present invention can be administered separately byenteral or parenteral administration methods or in combination withother suitable compounds to effect the desired treatment goals.

The pharmaceutically acceptable carrier and/or diluent must be ofsufficiently high purity and of sufficiently low toxicity to render itsuitable for administration to the human subject being treated. Itfurther should maintain or increase the stability of the therapeuticcomposition. The pharmaceutically acceptable carrier can be liquid orsolid and is selected, with the planned manner of administration inmind, to provide for the desired bulk, consistency, etc., when combinedwith other components of the therapeutic composition of the invention.For example, the pharmaceutically acceptable carrier can be, withoutlimitation, a binding agent (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calciumhydrogen phosphate, etc.), a lubricant (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.), a disintegrant (e.g., starch, sodiumstarch glycolate, etc.), or a wetting agent (e.g., sodium laurylsulfate, etc.). Other suitable pharmaceutically acceptable carriers forthe compositions of the present invention include, but are not limitedto, water, salt solutions, alcohols, polyethylene glycols, gelatins,amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins,hydroxymethylcelluloses, polyvinylpyrrolidones and the like.

Such carrier solutions also can contain buffers, diluents and othersuitable additives. The term “buffer” as used herein refers to asolution or liquid whose chemical makeup neutralizes acids or baseswithout a significant change in pH. Examples of buffers envisioned bythe invention include, but are not limited to, Dulbecco's phosphatebuffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W),normal/physiologic saline (0.9% NaCl).

These pharmaceutically acceptable carriers and/or diluents may bepresent in amounts sufficient to maintain a pH of the therapeuticcomposition of between about 3 and about 10. As such, the bufferingagent may be as much as about 5% on a weight to weight basis of thetotal composition. Electrolytes such as, but not limited to, sodiumchloride and potassium chloride may also be included in the therapeuticcomposition.

In one aspect, the pH of the therapeutic composition is in the rangefrom about 4 to about 10. Alternatively, the pH of the therapeuticcomposition is in the range from about 5 to about 9, from about 6 toabout 9, or from about 6.5 to about 8. In another embodiment, thetherapeutic composition comprises a buffer having a pH in one of said pHranges. In another embodiment, the therapeutic composition has a pH ofabout 7. Alternatively, the therapeutic composition has a pH in a rangefrom about 6.8 to about 7.4. In still another embodiment, thetherapeutic composition has a pH of about 7.4.

The sterile composition of the invention may be a sterile solution orsuspension in a nontoxic pharmaceutically acceptable medium. The term“suspension” as used herein may refer to non-adherent conditions inwhich cells are not attached to a solid support. For example, cellsmaintained in suspension may be stirred and are not adhered to asupport, such as a culture dish.

A suspension is a dispersion (mixture) in which a finely-divided speciesis combined with another species, with the former being so finelydivided and mixed that it doesn't rapidly settle out. A suspension maybe prepared using a vehicle such as a liquid medium, including asolution. In particular embodiments, the therapeutic composition of theinvention is a suspension, where the stem and/or progenitor cells aredispersed within an acceptable liquid medium or solution, e.g., salineor serum-free medium, and are not attached to a solid support. Ineveryday life, the most common suspensions are those of solids in liquidwater. Among the acceptable diluents, e.g., vehicles and solvents, thatmay be employed are water, Ringer's solution, isotonic sodium chloride(saline) solution, and serum-free cell culture medium. In someembodiments, hypertonic solutions are employed in making suspensions. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For parenteral application, particularly suitablevehicles consist of solutions, preferably oily or aqueous solutions, aswell as suspensions, emulsions, or implants. Aqueous suspensions maycontain substances which increase the viscosity of the suspension andinclude, for example, sodium carboxymethyl cellulose, sorbitol and/ordextran. In some embodiments, the infusion solution is isotonic tosubject tissues. In some embodiments, the infusion solution ishypertonic to subject tissues.

The pharmaceutically acceptable carrier, diluents, and other componentscomprising the administration-ready therapeutic composition of theinvention are derived from U.S. Pharmaceutical grade reagents that willpermit the therapeutic composition to be used in clinical regimens.Typically, these finished reagents, including any medium, solution, orother pharmaceutically acceptable carriers and/or diluents, aresterilized in a manner conventional in the art, such as filtersterilized, and are tested for various undesired contaminants, such asmycoplasma, endotoxin, or virus contamination, prior to use. Thepharmaceutically acceptable carrier in one embodiment is substantiallyfree of natural proteins of human or animal origin, and suitable forstoring the population of cells of the therapeutic composition,including hematopoietic stem and progenitor cells. The therapeuticcomposition is intended to be administered into a human patient, andthus is substantially free of cell culture components such as bovineserum albumin, horse serum, and fetal bovine serum.

The invention also contemplates, in part, the use of a pharmaceuticallyacceptable cell culture medium in particular compositions and/orcultures of the present invention. Such compositions are suitable foradministration to human subjects. Generally speaking, any medium thatsupports the maintenance, growth, and/or health of the desiredreprogrammed and/or programmed cells of the invention are suitable foruse as a pharmaceutical cell culture medium. In particular embodiments,the pharmaceutically acceptable cell culture medium is a serum freemedium.

The therapeutic composition may comprise serum-free medium suitable forstoring the population of cells comprising the composition. In variousembodiments, the serum-free medium is animal-free, and may optionally beprotein-free. Optionally, the medium may contain biopharmaceuticallyacceptable recombinant proteins. “Animal-free” medium refers to mediumwherein the components are derived from non-animal sources. Recombinantproteins replace native animal proteins in animal-free medium and thenutrients are obtained from synthetic, plant or microbial sources.Protein-free medium, in contrast, is defined as substantially free ofprotein.

The serum-free medium employed in the present invention is a formulationsuitable for use in human therapeutic protocols and products. Oneserum-free media is QBSF-60 (Quality Biological, Inc.), as described inU.S. Pat. No. 5,945,337. QBSF-60 isoptimized with U.S. Pharmaceuticalgrade components and is composed of the basal medium IMDM plus 2 mML-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, humaninjectable grade serum albumin (4 mg/ml) (Alpha TherapeuticCorporation), partially iron saturated human transferrin (300 μg/ml)(Sigma Chemical Corporation or Bayer Corporation) and human recombinantsodium insulin (0.48 U/ml) (Sigma). Other serum-free media known in theart include, but are not limited to: Life Technologies CatalogueStemPro-34 serum free culture media; Capmany, et al., Short-term,serum-free, static culture of cord blood-derived CD34⁺ cells: effects ofFLT3-L and MIP-1α on in vitro expansion of hematopoietic progenitorcells, Haematologica 84:675-682 (1999); Daley, J P, et al., Ex vivoexpansion of human hematopoietic progenitor cells in serum-freeStemPro™-34 Medium, Focus 18(3):62-67; Life Technologies Catalogueinformation on AIM V serum free culture media; BioWhittaker Catalogueinformation on X-VIVO 10 serum free culture media; U.S. Pat. No.5,397,706 entitled Serum-free basal and culture medium for hematopoieticand leukemia cells; no cell proliferation; Kurtzberg et al., 18:153-4(2000); Kurtzberg et al., Exp Hematol 26(4):288-98 (April 1998).

One having ordinary skill in the art would appreciate that the aboveexample of medium is illustrative and in no way limits the formulationof media suitable for use in the present invention and that there aremany such media known and available to those in the art.

In various embodiments, the therapeutic composition of the inventioncomprises a sterile solution of human serum albumin (HSA), such as 5%HSA, and low molecular weight (LMW) dextran.

The therapeutic composition is substantially free of mycoplasm,endotoxin, and microbial contamination. In particular embodiments, thetherapeutic composition contains less than about 10, 5, 4, 3, 2, 1, 0.1,0.05 μg/ml bovine serum albumin.

By “substantially free” with respect to endotoxin is meant that there isless endotoxin per dose of cells than is allowed by the FDA for abiologic, which is a total endotoxin of 5 EU/kg body weight per day,which for an average 70 kg person is 350 EU per total dose of cells.

With respect to mycoplasma and microbial contamination, “substantiallyfree” as used herein means a negative reading for the generally acceptedtests known to those skilled in the art. For example, mycoplasmacontamination is determined by subculturing a sample of the therapeuticcomposition in broth medium and distributed over agar plates on day 1,3, 7, and 14 at 37° C. with appropriate positive and negative controls.The sample appearance is compared microscopically, at 100×, to that ofthe positive and negative control. Additionally, inoculation of anindicator cell culture is incubated for 3 and 5 days and examined at600× for the presence of mycoplasmas by epifluorescence microscopy usinga DNA-binding fluorochrome. The sample is considered satisfactory if theagar and/or the broth media procedure and the indicator cell cultureprocedure show no evidence of mycoplasma contamination.

EXAMPLES Example 1 CXCR4 mRNA Expression Levels in Treated HSPCs

CXCR4 qPCR Using the Fluidigm Platform

Real-time PCR transcript quantitation of gene expression from ex vivotreated human umbilical cord blood derived CD34⁺ cells (Stem CellTechnologies, Vancouver, BC, Canada) was performed using the BioMarkDynamic Array microfluidics system (Fluidigm Corporation, South SanFrancisco, Calif., USA).

CD34⁺ cells derived from cord blood or mobilized peripheral blood weretreated in Serum-Free Expansion Medium (SFEM; e.g., StemSpan® fromStemCell Technologies, Inc.) for four hours at 37° C., 5% CO₂ with 10 uMprostaglandin pathway agonist alone or in combination with 10 uM of aglucocorticoid. Prostaglandin pathway agonists included 16,16-dimethylPGE₂ (dmPGE₂), 20-ethyl PGE2 (ePGE₂), 15(S)-15-methyl PGE2 (mPGE₂), andPGE₂. Glucocorticoids included hydrocortisone, dexamethasone, medrysone,alclometasone, or triamcinalone. After treatment, cells were washed withSFEM and centrifuged at 300 g for 10 minutes.

Total RNA was isolated then isolated from treated cells using Pico PureRNA Isolation Kit (Molecular Devices, Sunnyvale, Calif., USA).Complimentary DNA (cDNA) was reverse transcribed from 50 ng of isolatedtotal RNA using the High-Capacity cDNA Reverse Transcription Kit (LifeTechnologies Corporation, Carlsbad, Calif., USA).

cDNA was pre-amplified for specific target genes (96) using a 200 nMmixture of 96 gene specific Applied Biosystems TaqMan Assays (see Table1), including 3 reference control genes (GAPDH, HPRT1, and QARS) usingthe TaqMan PreAmp Master Mix Kit (Life Technologies) protocol. Specifictarget amplification (STA) from cDNA was performed using 14 cycles ofamplification with the standard cycling conditions using themanufacturer's protocol. For samples, the reaction mix contained 3.0 μLGene Expression Master Mix (Life Tech.), 0.3 μL Sample Loading Buffer(Fluidigm), 1.5 μL diluted (1:5 sterile nH2O) STA cDNA, and 1.2 μLsterile diH₂O for loading into the sample inlets of the 96.96 DynamicArray (Fluidigm).

Samples were run in replicates, from 5 to 9 wells. The reaction mixcontained 2.5 μL Gene Specific Taqman Assays (20×) and 2.5 μL AssayLoading Buffer (Fluidigm) for loading into the assay inlets on the 96.96Dynamic Array (Fluidigm). 96.96 Dynamic arrays were loaded using aNanoFlex IFC Controller HX (Fluidigm) and real-time reactions wereperformed using a BioMark Real-Time PCR System (Fluidigm).

Results were analyzed using BioMark Real-Time PCR Analysis software.Average Cts were calculated from the sample replicates and delta-deltaCts (AACt) were calculated using the mean of 3 reference genes (GAPDH,HPRT1, QARS) against a vehicle only sample. Cts above 28 were excludedfrom the calculations. The results were displayed in an Excel graphicbar graph (Microsoft Corp., Redmond, Wash., USA) showing average foldchange (2^ ΔΔCt) for CXCR4. Error bars depict +/− the Standard Deviation(SD) of the replicate measurements.

Results

An increase in CXCR4 mRNA expression was observed in umbilical cordblood CD34⁺ cells treated in SFEM with 10 μM 16,16-dimethyl PGE₂ (22fold), or a combination of 10 μM dmPGE₂ and 10 μM glucocorticoid (50 to61 fold), when compared to DMSO treated cells. A synergistic increase inCXCR4+ mRNA levels was detected following a combination treatment of 10μM dmPGE₂ and 10 μM of any one of five different glucocorticoids (FIG.1). Glucocorticoids act synergistically with dmPGE2 to increase CXCR4gene expression.

A similar synergistic increase in CXCR4 mRNA expression was alsoobserved in umbilical cord blood CD34⁺ cells treated in SFEM for 4 hourswith other prostaglandin pathway agonists, 10 μM PGE₂ (8 to 61 foldincrease) (FIG. 2A), 10 μM 15,15-methyl PGE₂ (16 to 50 fold increase)(FIG. 2B), and 10 μM 20-ethyl PGE₂ (16 to 55 fold increase) (FIG. 2C)when combined with a glucocorticoid compared DMSO treated cells.Glucocorticoids also act synergistically with other prostaglandinpathway agonists to increase CXCR4 gene expression.

TABLE 1 Applied Biosystems TaqMan Assays Gene Assay ID Gene Assay IDGene Assay ID Gene Assay ID ANGPT1 Hs00375822_m1 CXCL6 Hs00237017_m1PROM1 Hs01009250_m1 BMP4 Hs00370078_m1 ANGPT2 Hs01048042_m1 IKBKBHs00233287_m1 PECAM1 Hs00169777_m1 TIE1 Hs00178500_m1 AREG Hs00950669_m1CASP3 Hs00234387_m1 JAG1 Hs01070032_m1 GAPDH Hs99999905_m1 ARNTHs00231048_m1 CREM Hs01590456_m1 CTGF Hs00170014_m1 CD40L Hs00163934_m1BAX Hs00180269_m1 HGF Hs00300159_m1 SOD2 Hs00167309_m1 PDGFBHs00966522_m1 THBS1 Hs00962908_m1 DUSP4 Hs01027785_m1 CYR61Hs00155479_m1 CXCL1 Hs00236937_m1 TEK Hs00945146_m1 CFLAR Hs01116280_m1IGF2 Hs00171254_m1 CXCR4 Hs00976734_m1 MMP2 Hs01548727_m1 FGF2Hs00266645_m1 PTGS2 Hs00153133_m1 RAC2 Hs01032884_m1 PDGFR Hs01019589_m1NR4A2 Hs00428691_m1 TERT Hs00972656_m1 TGFB1 Hs00998133_m1 MMP9Hs00234579_m1 CD40 Hs00374176_m1 CD44 Hs01075861_m1 HMGB1 Hs01923466_g1NOS3 Hs01574659_m1 KDR Hs00911700_m1 ITGB1 Hs00559595_m1 CTNNB1Hs00170025_m1 CSF3 Hs00357085_g1 IL8 Hs00174103_m1 PLAUR Hs00182181_m1DUSP4 Hs00175210_m1 BCL2 Hs00608023_m1 BMP2 Hs00154192_m1 CSF1Hs00174164_m1 AKT1 Hs00178289_m1 VEGFA Hs00900055_m1 ICAM1 Hs00164932_m1CXCL3 Hs00171061_m1 CASP8 Hs01018151_m1 CD34 Hs00990732_m1 IL1AHs00174092_m1 CD47 Hs00179953_m1 CCL7 Hs00171147_m1 HIF1A Hs00936371_m1EDN1 Hs00174961_m1 S1PR1 Hs00173499_m1 CCR1 Hs00174298_m1 SMAD4Hs00929647_m1 FLT1 Hs01052961_m1 GEM Hs00738924_m1 CD151 Hs00388381_m1PGF Hs01119262_m1 NFKB1 Hs00765730_m1 SMAD2 Hs00183425_m1 CXCR7Hs00604567_m1 TGFB3 Hs01086000_m1 CXCL5 Hs00171085_m1 CCND1Hs00765553_m1 HBEGF Hs00181813_m1 NR3C1 Hs00353740_m1 TNF Hs00174128_m1ITGAL Hs00158218_m1 CXCR2 Hs01011557_m1 STAT1 Hs01013996_m1 ITGA4Hs00168433_m1 LIF Hs00171455_m1 RASA1 Hs00243115_m1 CDH5 Hs00901463_m1HPRT1 Hs01003267_m1 EFNB2 Hs00187950_m1 RGS16 Hs00161399_m1 CXCL2Hs00601975_m1 ITGA5 Hs01547673_m1 CXCL12 Hs00171022_m1 TIMP1Hs00171558_m1 FOSL2 Hs00232013_m1 ITGB2 Hs00164957_m1 QARS Hs00192530_m1TIMP2 Hs00234278_m1CXCR4 qPCR Using the Applied Biosystems StepOnePlus

Real-time PCR transcript quantitation from ex vivo treated cord CD34⁺cells (Stem Cell Technologies, Vancouver, BC, Canada) and mobilizedperipheral blood CD34⁺ cells (All Cells, LLC, Emoryville, Calif., USA)was performed using the Applied Biosystems StepOne Plus system (LifeTechnologies Corporation, Carlsbad, Calif., USA) (see FIG. 3). CD34⁺cells were treated in SFEM for four hours at 37° C., 5% CO₂ with 10 uM16,16-dimethyl PGE₂ (dmPGE₂) alone or in combination with 10 uMmedrysone. After treatment, cells were washed with SFEM and centrifugedat 300 g for 10 minutes.

Total RNA was then isolated from treated cells using Pico Pure RNAIsolation Kit (Molecular Devices, Sunnyvale, Calif., USA). ComplimentaryDNA (cDNA) was reverse transcribed from 50 ng of isolated total RNAusing the High-Capacity cDNA Reverse Transcription Kit (LifeTechnologies).

Real-Time PCR analysis was run on the cDNA samples in duplicate. Thereaction mix contained 1 μL Gene Specific Taqman Assay (20×) and 10 μLGene Expression Master Mix (Life Technologies) with the remaining volumeof 9 μL contained 10 ng cDNA and sterile diH₂O.

CXCR4 results (assay from Table 1) were analyzed using AppliedBiosystems StepOne Software v2.1 Analysis software. Average Cts werecalculated from the sample replicates and delta-delta Cts (ΔΔCt) werecalculated using GAPDH results (assay from Table 1) as a reference geneagainst a vehicle only sample. Results were displayed in an Excelgraphic bar graph (Microsoft Corp., Redmond, Wash., USA) showing averagefold change (2^ ΔΔCt) for CXCR4. Error bars depict +/− the StandardDeviation (SD) of the replicate measurements (See FIGS. 1-3, 5, and 6).

Results

A similar increase in CXCR4 mRNA expression was observed in both cordblood derived CD34⁺ cells and CD34⁺ cells isolated from mobilizedperipheral blood (22 and 27 fold increase respectively) after a 4 hourtreatment in SFEM with 10 μM 16,16-dimethyl PGE₂. A similar synergisticincrease in CXCR4 mRNA was detected in both cord blood derived CD34⁺cells and CD34⁺ cells isolated from mobilized peripheral blood (60 and59 fold respectively) after a 4 hour combination treatment in SFEM witha prostaglandin pathway agonist (dmPGE₂) and a glucocorticoid(medrysone) (FIG. 3). CD34⁺ cells respond similarly to either treatmentregardless of source of origin.

Example 2 Treatment of CD34⁺ Cells with a Combination of dmPGE₂ andMedrysone Results in Increased CXCR4 Surface Protein Expression

CXCR4 Surface Expression Analysis on frozen CD34^(+CB and PB)

CD34⁺ cord blood (CB) cells (Stem Cell Technologies) and CD34⁺ mobilizedperipheral blood (mPB) cells (All Cells) were treated in SFEM for 2 and4 hours at 37° C., 5% CO₂ with 10 μM dmPGE₂, dmPGE₂ and medrysone, orDMSO as control. After treatment, cells were washed with SFEM andcentrifuged at 300 g for 10 minutes. Cells were then resuspended in SFEMfor incubation at 37° C. 5% CO₂ for diverse amount of time.

mPB and CB CD34⁺ cells were treated in SFEM for 2 or 4 hours withdmPGE₂, dmPGE₂ and medrysone, or DMSO, and cells were then assessed forCXCR4 surface protein expression at different time points during andafter treatment (Table 2). In order to measure CXCR4 levels treatedcells were centrifuged at 300 g for 10 minutes and resuspended instaining media containing Lineage cocktail 1-FITC, CD34-APC,CXCR4(CD184)-PE, and incubated on ice for 20 minutes. Fresh stainingmedia was then added to the cells to wash the cells from any residualunbound antibodies, the cells were centrifuged at 300 g for 10 minutes,and this washing procedure was repeated twice. The stained cells wereacquired on a Guava EasyCyte 8HT flow cytometer and analysis wasperformed using FloJo Software Package (Treestar).

TABLE 2 Treatments and time points used for CXCR4 protein surfaceanalysis Time Treatments 2 hr Tx DMSO 10 uM 10 uM dmPGE2 + 2 hr Tx + 2hr at 37° C. dmPGE2 10 uM Medrysone 2 hr Tx + 4 hr at 37° C. 4 hr Tx 4hr Tx + 2 hr at 37° C. 4 hr Tx + 4 hr at 37° C.Results

An increase in CXCR4 RNA expression was observed in CD34⁺ cells fromcord blood or mPB cells treated in SFEM with 10 μM 16,16-dimethyl PGE₂and dmPGE₂ and medrysone for 2 and 4 hours at 37° C. when compared toDMSO treated cells. The highest percentage of CXCR4+ cells was obtained2 hours after a 4 hour treatment with dmPGE₂ and medrysone (FIGS. 5 and6) for both type of cells regardless of the cell source. For mobilizedperipheral blood CD34⁺ cells, 75% of the cells expressed CXCR4+ comparedto 8% for control (FIG. 5). For cord blood CD34⁺ cells, 25% of the cellsexpressed CXCR4 after dmPGE₂ and medrysone treatment compared to 3-6%for control samples (FIG. 6).

CD34⁺ cells from human bone marrow were also tested for their ability torespond to 16,16-dimethyl PGE₂ and the combination of dmPGE₂ andmedrysone. In this case, previously frozen bone marrow CD34⁺ weretreated in SFEM with 10 μM 16,16-dimethyl PGE₂+10 μM medrysone for 4hours at 37° C. i. Cells were then washed and resuspended in SFEM for 2hours. The CXCR4 surface protein was then assessed by flow cytometry aspreviously described. Treatment of BM CD34⁺ cells in SFEM with 10 μM16,16-dimethyl PGE₂ alone or 10 μM 16,16-dimethyl PGE₂+10 μM medrysoneresults in an increase in the level of CXCR4 protein expression of 12and 20 fold increase, respectively. Furthermore, treatment in SFEM with10 μM 16,16-dimethyl PGE₂+10 μM medrysone results in a 12-fold increasein the percentage of CXCR4⁺ cells (FIGS. 4A and 4B).

Example 3 Sdf-1 Transwell Migration Assays

Methods

Transwell migration assays were performed using 96-well chemotaxischambers, 5 μM pore size polycarbonate membrane (Corning Inc., Corning,N.Y.) in accordance with manufacturer's instructions. Briefly, CD34⁺cells were then treated for 4 hours at 37° C. with 16,16-dimethyl PGE₂(dmPGE₂), dmPGE₂ and glucocorticoid, or DMSO control at a concentrationof 10 μM in StemSpan® media (Stem Cell Technology, Vancouver, Canada).The cells were then washed by centrifugation (300×g for 10 minutes) andresuspended in transwell assay buffer (Phenol Red Free RPMI media(Mediatech), 0.5% lipid free BSA (Sigma-Aldrich) at a concentration of40,000-60,000 cells/75 μl.

To test the duration of the treatment effects, one portion of treatedcells was washed by centrifugation (300×g for 10 minutes) andresuspended in StemSpan® media for 4 hours at 37° C. without dmPGE₂,glucocorticoids, or DMSO and then washed again by centrifugation (300×gfor 10 minutes) and resuspended in transwell assay buffer (Phenol RedFree RPMI media (Mediatech), 0.5% lipid free BSA (Sigma-Aldrich) at aconcentration of 40,000-60,000 cells/75 μl.

Seventy-five μl of cell suspension was added to the upper chamber of theplate, while 235 μl of transwell assay media containing 0 or 50 ng/mlSDF1α (R&D system, Minneapolis, Minn.) was added to the bottom well.Total cell number in the lower well was obtained by flow cytometry after2.5 hours of incubation at 37° C., 5% CO₂.

Results

CD34⁺ cells were treated with DMSO control, dmPGE₂, or dmPGE₂ andmedrysone as described above. The treated cells were placed in the upperchambers of a transwell culture plate with 0 ng/mL SDF1 or 50 ng/mL SDF1in the lower chambers. Migration was expressed as the % of cells added,i.e., the number of cells in the lower chamber normalized to the numberof cells initially added to the upper chamber. dmPGE₂ treatmentincreased SDF1-driven migration compared to the DMSO control (See FIG.7). The combination treatment of dmPGE₂ and medrysone increasedSDF1-driven cell migration more than dmPGE₂ alone or DMSO control (SeeFIG. 7). Thus, CD34⁺ cells in SFEM treated with dmPGE₂, or dmPGE₂ andmedrysone migrated more efficiently towards SDF1 compared to DMSOcontrol treated cells.

CD34⁺ cells were treated with DMSO control, dmPGE₂, or dmPGE₂ and aglucosteroid (medrysone, hydrocortisone, triamcinolone, alclometasone,alclometasone dipropionate, or dexamethasone) as described above. Thetreated cells were placed in the upper chambers of a transwell cultureplate with 0 ng/mL SDF1 or 50 ng/mL SDF1 in the lower chambers.Migration was expressed as the % of cells added, i.e., the number ofcells in the lower chamber normalized to the number of cells initiallyadded to the upper chamber. dmPGE₂ treatment increased SDF1-driven cellmigration compared to the DMSO control (See FIG. 8). Moreover, treatmentwith dmPGE2 combined with either medrysone, hydrocortisone,triamcinolone, alclometasone, alclometasone dipropionate, ordexamethasone increased SDF1-driven cell migration more effectively thandmPGE2 alone or DMSO control (See FIG. 8). Thus, CD34⁺ cells treated inSFEM with dmPGE₂, or dmPGE₂ and various glucocorticoids migrated moreefficiently towards SDF1 compared to DMSO control treated cells andshowed that the enhanced migration property of the prostaglandin pathwayagonist/glucocorticoid treated cells is not limited to a particularglucocorticoid.

The duration of the enhanced migration effect of dmPGE₂/glucocorticoidtreated cells towards SDF-1 was tested. CD34⁺ cells were treated withDMSO or dmPGE₂ and medrysone. Freshly treated cells or treated cellsincubated for an additional 4 without further treatment (as describedabove) were placed in the upper chambers of a transwell culture platewith 0 ng/mL SDF1 or 50 ng/mL SDF-1 in the lower chambers. Migration wasexpressed as the % of cells added, i.e., the number of cells in thelower chamber normalized to the number of cells initially added to theupper chamber. dmPGE₂ and medrysone treatment increased SDF1-driven cellmigration compared to the DMSO control (See FIG. 9). Moreover, dmPGE₂and medrysone treated cells incubated for an additional 4 hours with nofurther treatment migrated as well as the freshly treated cells. Thus,the enhanced migration effect of prostaglandin pathwayagonist/glucocorticoid treated cells towards SDF-1 is stable for atleast four hours, an indicated that the effect would also be present inadministering the treated cells to a subject.

Example 4 PGE₂ and PGE₂/Glucocorticoid Treated CD34+ Cells ImproveNeurological and Locomotor Function in a Rat Ischemia Model

Methods

Adult male Wistar rats were subjected to a transient focal ischemia byblocking the right middle cerebral artery MCAO model (Middle CerebralArtery Occlusion). A surgical nylon suture with a rounded tip wasadvanced from the external carotid artery into the lumen of the internalcarotid artery until it blocked the origin of the middle cerebralartery. After 2 hours, the suture was withdrawn to allow reperfusion.One day after reperfusion, rats were injected via tail vein with eitherHanks Balanced Salt Solution (HBSS), DMSO-treated CD34⁺ cells, or CD34⁺cells treated with dmPGE₂ and medrysone. A phospodiesterase type 4inhibitor (YM976) was also included to increase the durability of theenhanced cell effect. Our work demonstrates that PDE4 inhibitors do notsignificantly change the properties of the enhanced cell. Cells wereincubated with compound or DMSO in culture medium for 4 hours at 37° C.Before injection, pretreated cells were centrifuged; the resultantsupernatant was aspirated; and the cell pellet was resuspended in HBSS.

One day and 1, 2, 3, 4 and 5 weeks after injection, rats were assessedfor neurological deficits with behavioral testing performed by aninvestigator who was blinded to the experimental groups. A modifiedNeurological Severity Score (mNSS) was calculated based on a publishedpanel of motor, sensory, balance and reflex tests (Chen et al., Stroke32:2682-2688 (2001)).

In addition, 1 day and 1, 2, 3, 4 and 5 weeks after injection, locomotorfunction was evaluated in the treated rats with a foot-fault test inwhich the animal crossed a perforated walkway. The total number offorelimb steps and the number of missteps, in which the left forelimbfell through a perforation, were measured.

Results

Rats were administered treated HSPCs, and the ability of the treatmenteffect to reduce neurological deficit in the MCAO stroke model wastested. Treated HSPCs were intravenously injected 24 hours afterunilateral ischemic brain injury. Neurological function was assessedwith a battery of behavioral tests and reported as mNSS. Cells treatedwith dmPGE₂ and medrysone significantly improved mNSS at 7, 14 and 35days compared to vehicle control, while DMSO-treated cells did notsignificantly affect mNSS (See FIG. 10). *p<0.05 (n=6/group).

Rats were administered HSPCs treated with dmPGE₂ and medrysone, and theability of the treatment effect to reduce locomotor deficit in the MCAOstroke model was tested. Treated HSPCs were intravenously injected 24hours after unilateral ischemic brain injury. Locomotor function wasassessed as % foot-faults when crossing a perforated walkway. Cellstreated with dmPGE₂ and medrysone significantly decreased % foot-faultsat 7 and 35 days compared to vehicle control, while DMSO-treated cellsdid not significantly affect % foot-faults (See FIG. 11). *p<0.05(n=6/group).

Thus, the HSPCs treated with a prostaglandin pathway antagonist and aglucocorticoid effectively treated ischemia and the symptoms associatedtherewith, in the rat MCAO model.

Example 5 Methods

Isolation of Lin(−) CD34⁺ Cells from Treated Whole Cord Blood

Human whole cord blood mononuclear cells were obtained from Stem CellTechnologies (Vancouver, Canada). Upon thawing, the cells were treatedwith 16,16-dimethyl PGE₂ or appropriate controls, e.g., DMSO, in LMD/5%HSA medium.

After treatment, the cells were washed with LMD/5% HSA medium,centrifuged for 10 minutes at 650×g at room temperature and resuspendedin a cold selection buffer (phosphate buffered saline (PBS) with no Ca⁺or Mg⁺; 2 mM EDTA; and 0.5% HSA). Magnetic selection was performed usingthe Lineage (Lin) Depletion Kit (Miltenyi Biotec, CA) followed by aCD34⁺ enrichment kit (Miltenyi Biotec). Lineage depletion and CD34⁺ cellenrichment were performed according to manufacturer's instructions usinga QuadroMACS™ separator. During this process, the cells were kept at 4°C. Once the Lin-CD34⁺ cells were isolated from the treated whole cordblood, an aliquot was analyzed by flow cytometry to assess purity.Purity of the cells was greater than 90%. The majority of the cells wereused for RNA extraction using the Pico Pure RNA Isolation Kit (MolecularDevices, Sunnyvale, Calif.) for Affymetrix analysis.

The CD34⁺ cells described in the above examples were isolated from cordblood, mobilized peripheral blood and bone marrow cells, as noted, andobtained from Stem Cell Technologies and All Cells LLC. Upon receivingthese cells, the level of differentiated cells contamination wasdetermined by flow cytometry, based on the amount of lineage markerspresent on the surface of the CD34⁺ cells. CD34⁺ cells expressinglineage markers are differentiated progenitor cells that do not have thesame self-renewal capacity as lineage negative CD34⁺ cells. All CD34⁺cells obtained from these companies and referenced in the experimentsherein were at least 85% CD34⁺/Lin(−) cells.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The invention claimed is:
 1. A human hematopoietic stem or progenitorcell that has been contacted ex vivo with one or more agents thatincrease CXCR4 gene expression in the cell by at least about 30 fold inthe contacted hematopoietic stem or progenitor cell compared to ahematopoietic stem or progenitor cell that has not been contacted exvivo with one or more agents that increase CXCR4 gene expression.
 2. Thehematopoietic stem or progenitor cell of claim 1, wherein the one ormore agents comprise (i) one or more prostaglandin pathway agonists; and(ii) one or more glucocorticoids.
 3. The hematopoietic stem orprogenitor cell of claim 2, wherein the one or more prostaglandinpathway agonists comprises PGE₂ or a PGE₂ analogue or derivativethereof.
 4. The hematopoietic stem or progenitor cell of claim 2,wherein the one or more glucocorticoids is selected from the groupconsisting of medrysone, alclometasone, alclometasone dipropionate,amcinonide, beclometasone, beclomethasone dipropionate, betamethasone,betamethasone benzoate, betamethasone valerate, budesonide, ciclesonide,clobetasol, clobetasol butyrate, clobetasol propionate, clobetasone,clocortolone, cloprednol, cortisol, cortisone, cortivazol, deflazacort,desonide, desoximetasone, desoxycortone, desoxymethasone, dexamethasone,diflorasone, diflorasone diacetate, diflucortolone, diflucortolonevalerate, difluorocortolone, difluprednate, fluclorolone, flucloroloneacetonide, fludroxycortide, flumetasone, flumethasone, flumethasonepivalate, flunisolide, flunisolide hemihydrate, fluocinolone,fluocinolone acetonide, fluocinonide, fluocortin, fluocoritin butyl,fluocortolone, fluorocortisone, fluorometholone, fluperolone,fluprednidene, fluprednidene acetate, fluprednisolone, fluticasone,fluticasone propionate, formocortal, halcinonide, halometasone,hydrocortisone, hydrocortisone acetate, hydrocortisone aceponate,hydrocortisone buteprate, hydrocortisone butyrate, loteprednol,meprednisone, 6amethylprednisolone, methylprednisolone,methylprednisolone acetate, methylprednisolone aceponate, mometasone,mometasone furoate, mometasone furoate monohydrate, paramethasone,prednicarbate, prednisolone, prednisone, prednylidene, rimexolone,tixocortol, triamcinolone, triamcinolone acetonide and ulobetasol. 5.The hematopoietic stem or progenitor cell of claim 1, wherein the cellis obtained from bone marrow, umbilical cord blood, mobilized peripheralblood, Wharton's jelly, placenta, or fetal blood.
 6. A therapeuticcomposition comprising a population of cells comprising humanhematopoietic stem or progenitor cells contacted ex vivo with one ormore agents under conditions sufficient to increase CXCR4 expression inthe human hematopoietic stem or progenitor cells by at least about 30fold compared to hematopoietic stem or progenitor cells that have notbeen contacted ex vivo with one or more agents that increase CXCR4 geneexpression.
 7. The therapeutic composition of claim 6, wherein the oneor more agents comprise (i) one or more prostaglandin pathway agonistsand (ii) one or more glucocorticoids.
 8. The therapeutic composition ofclaim 6, wherein gene expression of CXCR4 is increased by at least about40 fold in the contacted hematopoietic stem or progenitor cells comparedto hematopoietic stem or progenitor cells that have not been contactedex vivo with one or more agents that increase CXCR4 gene expression. 9.The therapeutic composition of claim 7, wherein the one or moreprostaglandin pathway agonists comprises PGE₂ or a PGE₂ analogue orderivative thereof.
 10. The therapeutic composition of claim 7, whereinthe one or more glucocorticoids is selected from the group consisting ofmedrysone, alclometasone, alclometasone dipropionate, amcinonide,beclometasone, beclomethasone dipropionate, betamethasone, betamethasonebenzoate, betamethasone valerate, budesonide, ciclesonide, clobetasol,clobetasol butyrate, clobetasol propionate, clobetasone, clocortolone,cloprednol, cortisol, cortisone, cortivazol, deflazacort, desonide,desoximetasone, desoxycortone, desoxymethasone, dexamethasone,diflorasone, diflorasone diacetate, diflucortolone, diflucortolonevalerate, difluorocortolone, difluprednate, fluclorolone, flucloroloneacetonide, fludroxycortide, flumetasone, flumethasone, flumethasonepivalate, flunisolide, flunisolide hemihydrate, fluocinolone,fluocinolone acetonide, fluocinonide, fluocortin, fluocoritin butyl,fluocortolone, fluorocortisone, fluorometholone, fluperolone,fluprednidene, fluprednidene acetate, fluprednisolone, fluticasone,fluticasone propionate, formocortal, halcinonide, halometasone,hydrocortisone, hydrocortisone acetate, hydrocortisone aceponate,hydrocortisone buteprate, hydrocortisone butyrate, loteprednol,meprednisone, 6a-methylprednisolone, methylprednisolone,methylprednisolone acetate, methylprednisolone aceponate, mometasone,mometasone furoate, mometasone furoate monohydrate, paramethasone,prednicarbate, prednisolone, prednisone, prednylidene, rimexolone,tixocortol, triamcinolone, triamcinolone acetonide and ulobetasol. 11.The therapeutic composition of claim 6, wherein the hematopoietic stemor progenitor cells have been contacted for a time of at least about onehour with at least one of (i) one or more prostaglandin pathway agonistsand (ii) one or more glucocorticoids.
 12. The therapeutic composition ofclaim 6, wherein the population of cells comprises at least about 0.01%and no more than about 50% of CD34+ cells.
 13. The therapeuticcomposition of claim 6, wherein the composition is not cultured.
 14. Amethod of increasing hematopoietic stem or progenitor cell homing,engraftment, or reconstitution in a subject comprising administering tothe subject a composition comprising a population of cells comprisinghuman hematopoietic stem or progenitor cells wherein: a) thehematopoietic stem or progenitor cells have been contacted ex vivo withone or more agents that increase CXCR4 gene expression in the cells; andb) gene expression of CXCR4 is increased at least about 30 fold in thecontacted hematopoietic stem or progenitor cells compared tohematopoietic stem or progenitor cells that have not been contacted exvivo with one or more agents that increase CXCR4 gene expression in thecells.
 15. The method of claim 14, wherein the one or more agentscomprise (i) one or more prostaglandin pathway agonists; and (ii) one ormore glucocorticoids.
 16. The method of claim 14, wherein thehematopoietic stem or progenitor cells are obtained from bone marrow,umbilical cord blood, mobilized peripheral blood, Wharton's jelly,placenta, or fetal blood.
 17. The method of claim 15, wherein geneexpression of CXCR4 is increased by at least about 40 fold in thecontacted hematopoietic stem or progenitor cells compared tohematopoietic stem or progenitor cells that have not been contacted exvivo with one or more agents that increase CXCR4 gene expression. 18.The method of claim 15, wherein the prostaglandin pathway agonistcomprises PGE₂ or a PGE2 analogue or derivative thereof.
 19. The methodof claim 15, wherein the one or more glucocorticoids are selected fromthe group consisting of medrysone, alclometasone, alclometasonedipropionate, amcinonide, beclometasone, beclomethasone dipropionate,betamethasone, betamethasone benzoate, betamethasone valerate,budesonide, ciclesonide, clobetasol, clobetasol butyrate, clobetasolpropionate, clobetasone, clocortolone, cloprednol, cortisol, cortisone,cortivazol, deflazacort, desonide, desoximetasone, desoxycortone,desoxymethasone, dexamethasone, diflorasone, diflorasone diacetate,diflucortolone, diflucortolone valerate, difluorocortolone,difluprednate, fluclorolone, fluclorolone acetonide, fludroxycortide,flumetasone, flumethasone, flumethasone pivalate, flunisolide,flunisolide hemihydrate, fluocinolone, fluocinolone acetonide,fluocinonide, fluocortin, fluocoritin butyl, fluocortolone,fluorocortisone, fluorometholone, fluperolone, fluprednidene,fluprednidene acetate, fluprednisolone, fluticasone, fluticasonepropionate, formocortal, halcinonide, halometasone, hydrocortisone,hydrocortisone acetate, hydrocortisone aceponate, hydrocortisonebuteprate, hydrocortisone butyrate, loteprednol, meprednisone,6a-methylprednisolone, methylprednisolone, methylprednisolone acetate,methylprednisolone aceponate, mometasone, mometasone furoate, mometasonefuroate monohydrate, paramethasone, prednicarbate, prednisolone,prednisone, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide and ulobetasol.
 20. The method of claim 15,wherein the hematopoietic stem or progenitor cells have been contactedwith one or more prostaglandin pathway agonists and one or moreglucocorticoids for a time of at least about one hour.
 21. The methodclaim 15, wherein the population of cells comprises at least about 0.01%and no more than about 50% of CD34+ cells.
 22. The method of claim 15,wherein the composition is not cultured.
 23. The method of claim 15,wherein the subject: a) has acute myelogenous leukemia (AML), acutelymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML),chronic lymphocytic leukemia (CLL), juvenile myelomonocytic leukemia,Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, severeaplastic anemia, Fanconi's anemia, paroxysmal nocturnal hemoglobinuria(PNH), pure red cell aplasia, amegakaryocytosis/congenitalthrombocytopenia, severe combined immunodeficiency syndrome (SCID),Wiskott-Aldrich syndrome, beta-thalassemia major, sickle cell disease,Hurler's syndrome, adrenoleukodystrophy, metachromatic leukodystrophy,myelodysplasia, refractory anemia, chronic myelomonocytic leukemia,agnogenic myeloid metaplasia, familial erythrophagocyticlymphohistiocytosis, or solid tumors; b) has breast cancer, ovariancancer, brain cancer, prostate cancer, lung cancer, colon cancer, skincancer, liver cancer, pancreatic cancer, or sarcoma; c) has receivedbone marrow ablative or non-myeolablative chemotherapy or radiationtherapy; or d) is a bone marrow donor.
 24. The hematopoietic stem orprogenitor cell of claim 1, wherein the cell is contacted with the oneor more agents at a temperature of about 22° C. to about 39° C.
 25. Thetherapeutic composition of claim 1, wherein the cell is contacted withthe one or more agents at a temperature of about 35° C. to about 39° C.26. The therapeutic composition of claim 6, wherein the cell iscontacted with the one or more agents at a temperature of about 22° C.to about 39° C.
 27. The therapeutic composition of claim 6, wherein thecell is contacted with the one or more agents at a temperature of about35° C. to about 39° C.
 28. The therapeutic composition of claim 6,wherein the population of cells is not expanded ex vivo.
 29. A humanhematopoietic stem or progenitor cell having CXCR4 gene expression of atleast 30-fold greater compared to basal CXCR4 gene expression of acontrol hematopoietic stem or progenitor cell.
 30. The humanhematopoietic stem or progenitor cell of claim 29, wherein thehematopoietic stem or progenitor cell has at least 40-fold greater CXCR4gene expression compared to basal CXCR4 gene expression.
 31. Apopulation of cells comprising the human hematopoietic stem orprogenitor cell of claim
 29. 32. The population of cells of claim 31,wherein the population comprises at least about 0.01% and no more thanabout 50% CD34+ cells.
 33. The population of cells of claim 31, whereinthe cells are ready for administration to a human subject in needthereof.
 34. A therapeutic composition comprising the humanhematopoietic stem or progenitor cell of claim
 29. 35. The hematopoieticstem or progenitor cell of claim 1, wherein the one or moreprostaglandin pathway agonists comprises 16,16-dimethyl-PGE₂.
 36. Thetherapeutic composition of claim 9, wherein the one or moreprostaglandin pathway agonists comprises 16,16-dimethyl-PGE₂.
 37. Thetherapeutic composition according to claim 11, wherein the one or moreprostaglandin pathway agonists comprises 16,16-dimethyl-PGE₂.
 38. Themethod of claim 15, wherein the one or more prostaglandin pathwayagonists comprises 16,16-dimethyl-PGE₂.
 39. The therapeutic compositionof claim 6, wherein the hematopoietic stem or progenitor cells have beencontacted for a time of two hours to six hours with at least one of (i)one or more prostaglandin pathway agonists and (ii) one or moreglucocorticoids.
 40. The method of claim 15, wherein the hematopoieticstem or progenitor cells have been contacted with one or moreprostaglandin pathway agonists and one or more glucocorticoids for atime of two hours to six hours.